Eribulin antibody-drug conjugates and methods of use

ABSTRACT

Antibodies, antigen-binding fragments, and conjugates (e.g., antibody-drug conjugates such as those comprising eribulin) thereof that bind to mesothelin are disclosed. The disclosure further relates to methods and compositions for use in the treatment of cancer by administering the compositions provided herein.

The present disclosure claims the benefit of priority to U.S.Provisional Patent Application No. 62/932,373, filed Nov. 7, 2019, whichis incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 28, 2020, isnamed 08061_0043-00000_SL.txt and is 36,541 bytes in size.

The present disclosure relates to anti-mesothelin antibodies andantigen-binding fragments thereof, as well as conjugates such asantibody-drug conjugates (ADCs), e.g., those comprising eribulin, andtheir use in the treatment and diagnosis of cancers that expressmesothelin and/or are amenable to treatment by disrupting tubulin or byadministering a composition disclosed herein.

Cancer is among the leading causes of morbidity and mortality worldwide,with approximately 14 million new cases and 8.2 million cancer-relateddeaths in 2012. The most common causes of cancer death are cancers of:lung (1.59 million deaths); liver (745,000 deaths); stomach (723,000deaths); colorectal (694,000 deaths); breast (521,000 deaths); andesophagus (400,000 deaths). The number of new cancer cases is expectedto rise by about 70% over the next two decades, to approximately 22million new cancer cases per year (World Cancer Report 2014).

Mesothelin, a glycosylphosphatidylinositol (GPI)-anchored cell surfaceprotein, is an attractive target for antibody-based cancer therapy dueto its high expression in various cancer types, including mesothelioma,ovarian cancer, and pancreatic adenocarcinomas (Tang et al. (2013)Anticancer Agents Med. Chem. 13(2):276-80). Although a fullunderstanding of the biological functions of mesothelin is lacking giventhat mesothelin knockout mice do not show any detectable phenotype, ithas been suggested that mesothelin plays a role in tumor adhesion andmetastasis (Bera and Pastan (2000) Mol. Cell Biol. 20(8):2902-6; Rump etal. (2004) J. Biol. Chem. 279(10):9190-8). Mesothelin is also believedto confer resistance to certain forms of chemotherapy, and to contributeto tumor progression by having a proliferative effect on cells(Bharadwaj et al. (2011) Mol. Cancer. 10:106; Li et al. (2008) Mol.Cancer Ther. 7(2):286-96).

Recent studies have shown that mesothelin may function as a masterregulator of epithelial-mesenchymal transition (EMT), a process closelyassociated with cancer metastasis and recurrence (He et al. (2017). MolCancer. 16:63). Without wishing to be bound by theory, it is believedthat inhibition of mesothelin, e.g., binding by an anti-mesothelinantibody, antigen-binding fragment, and/or ADC, may reduce EMT byinducing the reverse process, mesenchymal epithelial transition (MET),via suppression of TGF-β (transforming growth factor beta) signaling.Conversely, it is believed that overexpression of mesothelin may driveEMT through induction of cancer stem cell-like phenotypes associatedwith tumor progression and poor treatment response (He et al. (2017).Mol Cancer. 16:63; Koyama et al. (2017). J. Clin. Invest. 127(4):1254-1270).

A commonly encountered challenge in cancer therapy is that the limitedtherapeutic index of chemotherapeutics results in significant toxicityto normal tissues and thus limits their therapeutic utility. Oneapproach to achieve higher specificity for targeting cancer cells is byusing antibodies to deliver cytotoxic effects to cells expressingcertain tumor-specific antigens while sparing normal cells that expressmuch lower levels or none of such antigens (Awwad et al. Pharmaceutics(2018) 10(3); Lambert and Berkenblit (2018) 69: 191-207). Suchtumor-specific targeting can be exploited to both increase anti-tumoractivity and decrease off-target cytotoxicity of therapeutics.Antibodies targeting tumor-specific antigens may deliver cytotoxiceffects through a variety of mechanisms, including inhibiting thebiological activity of the antigen, eliciting an immune effectoractivity, and/or inducing antibody-dependent cellular cytotoxicity(Hendrinks et al. International Review of Cell and Molecular Biology(2017); Therapeutic Antibody Engineering (2012): 163-196, 459-595).

Selection of tumor-specific antigens for an antibody-based therapeuticapproach may involve specific expression of an antigen by tumor cellsand robust killing of the antigen-expressing tumor cells. Several humancancers have been found to express high levels of mesothelin, includinglung cancer, ovarian cancer, pancreatic cancer, and stomach cancer(Hassan et al. Eur. J. Cancer (2008) 44(1): 46-53; Hassan et al. J.Clin. Oncol. (2016) 34(34): 4171-4179). Mesothelin expression has alsobeen found in drug resistant cancers such as lung cancers with KRAS andSTK11 mutations with poor clinical response to checkpoint blockadeimmunotherapy, and HER2-negative gastric cancer. Additionally,correlation has been reported between mesothelin expression and theoverall survival of patients with lung adenocarcinoma and of patientswith gastric cancer metastasis, suggesting that high mesothelinexpression may be a predictor of worse clinical outcome (Kachala et al.(2014) Clin. Cancer. Res. 20(4): 1020-1028; Han et al. (2017) J. Pathol.Transl. Med. 51(2): 122-128). The prevalence of mesothelin expression inhuman cancers and its association with poor clinical outcome rendermesothelin a potential target for tumor antigen-specific drug deliveryapproaches, e.g., an antibody-mediated approach. Antibodies conjugatedwith cytotoxic compounds such as chemotherapeutics have also beenexplored to enhance the cell-killing activity of antibody-based drugdelivery to tumor cells. Nevertheless, the need remains to providesuitable antibodies and/or ADCs that offer a combination of efficienttumor targeting, on-target effects, bystander killing, and/or reducedoff-target effects.

Eribulin is a synthetic analog of the macrocyclic compound halichondrinB, which has been previously shown to be a potent inhibitor of tubulinpolymerization, microtubule assembly, and tubulin-depend GTP hydrolysis.Tubulin makes up dynamic filamentous cytoskeletal proteins calledmicrotubules that are involved in a variety of vital cellular functions,including intracellular migration and transport, cell signaling, themaintenance of cell shape, and cell division. The rapid dividing rate ofcancer cells makes them particularly sensitive to the obstruction oftubulin function. As such, halichondrin B and eribulin have demonstratednotable anti-cancer activities in vitro and in vivo (Tan et al. (2009)Clin Cancer Res. 15(12): 4213-4219; Vandat et al. (2009) J. Clin. Oncol.27(18): 2954-2961). The mesylate salt of eribulin (eribulin mesylate) iscurrently marketed under the trade name Halaven™ for the treatment ofpatients with refractory metastatic breast cancer.

While uses of eribulin have been reported in the art, including in theADC context, there remains a need to better deliver eribulin in atargeted fashion to particular tissues, e.g., cancer tissues thatexpress mesothelin. Likewise, there remains a need in the art forimproved antibodies that bind mesothelin with superior properties, e.g.,with respect to antigen-binding and/or the ability to effectivelydelivery payloads such as eribulin to a target cell or tissue expressingmesothelin.

In various embodiments, the present disclosure provides, in part, novelantibodies and antigen-binding fragments that may be used alone, linkedto one or more additional agents (e.g., as ADCs), or as part of a largermacromolecule (e.g., a bispecific antibody, multispecific antibody,alone or as a multispecific antibody linked to a payload in an ADCformat) and administered as part of pharmaceutical compositions orcombination therapies. In some embodiments, the antibodies orantigen-binding fragments are humanized. In some embodiments, theantibodies or antigen-binding fragments contain minimal sequencesderived from a non-human immunoglobulin and retain the reactivity of anon-human antibody while being less immunogenic in human. In certainembodiments, the antibodies and antigen-binding fragments may be usefulfor treating human cancer patients.

The present disclosure more specifically relates, in variousembodiments, to antibodies and antibody-drug conjugate compounds thatare capable of binding and/or killing tumor cells. In variousembodiments, the compounds are also capable of internalizing into atarget cell after binding. ADC compounds comprising a linker thatattaches an eribulin drug moiety to an antibody moiety are disclosed. Anantibody moiety may be a full-length antibody or an antigen-bindingfragment.

In some embodiments, an antibody or antigen-binding fragment disclosedherein comprises three heavy chain complementarity determining regions(HCDRs) comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ IDNO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3); and three light chaincomplementarity determining regions (LCDRs) comprising amino acidsequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO:6 (LCDR3), as defined by the Kabat numbering system (Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md. (1987 and 1991))); or three heavy chain complementaritydetermining regions (HCDRs) comprising amino acid sequences of SEQ IDNO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3); and threelight chain complementarity determining regions (LCDRs) comprising aminoacid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQID NO: 12 (LCDR3), as defined by the IMGT numbering system(International ImMunoGeneTics Information System (IMGT®)).

In some embodiments, an antibody or antigen-binding fragment disclosedherein comprises three heavy chain complementarity determining regions(HCDRs) from a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 13 and three light chain complementaritydetermining regions (LCDRs) from a light chain variable regioncomprising an amino acid sequence of SEQ ID NO: 14.

In some embodiments, an antibody or antigen-binding fragment disclosedherein is an anti-mesothelin antibody or antigen-binding fragment. Invarious embodiments, the antibody or antigen-binding fragment comprisesa heavy chain variable region comprising an amino acid sequence of SEQID NO: 13, and a light chain variable region comprising an amino acidsequence of SEQ ID NO: 14, or sequences that are at least 90% identicalto the disclosed sequences. In various embodiments, the antibody orantigen-binding fragment comprises a human IgG1 heavy chain constantregion comprising an amino acid sequence of SEQ ID NO: 15, and a humanIg kappa light chain constant region comprising an amino acid sequenceof SEQ ID NO: 16. In various embodiments, the antibody orantigen-binding fragment comprises a heavy chain amino acid sequence ofSEQ ID NO: 17, and a light chain amino acid sequence of SEQ ID NO: 18.

In various embodiments, an antibody or antigen-binding fragmentdisclosed herein comprises three heavy chain complementarity determiningregions (HCDRs) comprising amino acid sequences encoded by nucleic acidsequences of SEQ ID NO: 19 (HCDR1), SEQ ID NO: 20 (HCDR2), and SEQ IDNO: 21 (HCDR3); and three light chain complementarity determiningregions (LCDRs) comprising amino acid sequences encoded by nucleic acidsequences of SEQ ID NO: 22 (LCDR1), SEQ ID NO: 23 (LCDR2), and SEQ IDNO: 24 (LCDR3), as defined by the Kabat numbering system; or three heavychain complementarity determining regions (HCDRs) comprising amino acidsequences encoded by nucleic acid sequences of SEQ ID NO: 25 (HCDR1),SEQ ID NO: 26 (HCDR2), and SEQ ID NO: 27 (HCDR3); and three light chaincomplementarity determining regions (LCDRs) comprising amino acidsequences encoded by nucleic acid sequences of SEQ ID NO: 28 (LCDR1),SEQ ID NO: 29 (LCDR2), and SEQ ID NO: 30 (LCDR3), as defined by the IMGTnumbering system.

In various embodiments, the antibody or antigen-binding fragmentcomprises a heavy chain variable region comprising an amino acidsequence encoded by the nucleic acid sequence of SEQ ID NO: 31, and alight chain variable region comprising an amino acid sequence encoded bythe nucleic acid sequence of SEQ ID NO: 32. In various embodiments, theantibody or antigen-binding fragment comprises a heavy chain constantregion comprising an amino acid sequence encoded by the nucleic acidsequence of SEQ ID NO: 33, and a light chain constant region comprisingan amino acid sequence encoded by the nucleic acid sequence of SEQ IDNO: 34. In various embodiments, the antibody or antigen-binding fragmentcomprises a heavy chain comprising an amino acid sequence encoded by thenucleic acid sequence of SEQ ID NO: 35, and a light chain comprising anamino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:36.

In some embodiments, the antibody or antigen-binding fragment is afull-length antibody. In some embodiments, the antibody orantigen-binding fragment is a monospecific antibody or antigen-bindingfragment, a bispecific antibody or antigen-binding fragment, or amultispecific antibody or antigen-binding fragment. In some embodiments,the antibody or antigen-binding fragment is a single chain variablefragment (scFv), or a Fab fragment.

In various embodiments, the antibody or antigen-binding fragment isconjugated to a therapeutic agent, e.g., one or more small moleculesand/or additional antibodies or antigen-binding fragments. In someembodiments, the therapeutic agent is eribulin. In some embodiments, theantibody or antigen-binding fragment is 345A12-HC15-LC4.

In various embodiments, an ADC disclosed herein comprises Formula (I):Ab−(L−D)_(p)  (I)whereinAb is an antibody or antigen-binding fragment, wherein the antibody orantigen-binding fragment is capable of binding to mesothelin andcomprises three heavy chain complementarity determining regions (HCDRs)comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2(HCDR2), and SEQ ID NO: 3 (HCDR3); and three light chain complementaritydetermining regions (LCDRs) comprising amino acid sequences of SEQ IDNO: 4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as definedby the Kabat numbering system; or three heavy chain complementaritydetermining regions (HCDRs) comprising amino acid sequences of SEQ IDNO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3); and threelight chain complementarity determining regions (LCDRs) comprising aminoacid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQID NO: 12 (LCDR3), as defined by the IMGT numbering system;D is a therapeutic agent, e.g., an eribulin moiety;L is a cleavable linker that covalently attaches Ab to D; andp is an integer from 1 to 8.

In some embodiments, p is an integer from 1 to 6. In some embodiments, pis 2 or 6.

In some embodiments, the ADC comprises a cleavable linker comprising acleavable moiety that is positioned such that no part of the linker orthe antibody or antigen-binding fragment remains bound to thetherapeutic agent (e.g., eribulin) upon cleavage. In some embodiments,the cleavable linker comprises a cleavable peptide moiety that iscleavable by an enzyme such as cathepsin B. In some embodiments, thecleavable moiety comprises a cleavable peptide moiety, e.g., an aminoacid unit such as Val-Cit. In some embodiments, the amino acid unitcomprises valine-citrulline (Val-Cit).

In some embodiments, the cleavable linker comprises at least one spacerunit comprising at least one PEG moiety. In some embodiments, the spacerunit or linker comprises (PEG)₂. In some embodiments, a spacer unitattaches to the antibody moiety via a maleimide (Mal) moiety(“Mal-spacer unit”). In some embodiments, the Mal-spacer unit is joinedto the antibody or antigen-binding fragment via a cysteine residue onthe antibody or antigen-binding fragment (e.g., a LCcys80 residue on theantibody). In some embodiments, the Mal-spacer unit is joined to acysteine residue (e.g., LCcys80) of a light chain variable region on theantibody or antigen-binding fragment. In some embodiments, p is 2, suchthat two -L-D moieties are attached to the antibody or antigen-bindingfragment. In some embodiments, each -L-D moiety is attached to acysteine residue (e.g., LCcys80) of a light chain variable region on theantibody or antigen-binding fragment. In some embodiments, the cysteineresidue is a LCcys80, i.e., a cysteine residue at amino acid position 80of a light chain variable region on an antibody or an antigen-bindingfragment according to the Kabat numbering system. In some embodiments,the cleavable linker comprises the Mal-spacer unit and a cleavablepeptide moiety and the cleavable peptide moiety comprises Val-Cit. Insome embodiments, the Mal-spacer unit attaches the antibody orantigen-binding fragment to the cleavable moiety.

In some embodiments, the Mal-spacer unit comprises at least one PEGmoiety. In some embodiments, the linker comprises Mal-(PEG)₂. In someembodiments, the Mal-spacer unit attaches the antibody moiety to thecleavable moiety in the linker. In some embodiments, the cleavablemoiety in the linker is a cleavable peptide moiety, e.g., an amino acidunit. In some embodiments, the linker comprises Mal-(PEG)₂-Val-Cit.

In some embodiments, the cleavable moiety of the ADC is directly joinedto eribulin, or a spacer unit attaches the cleavable moiety in thelinker to the eribulin drug moiety and cleavage of the conjugatereleases eribulin from the antibody or antigen-binding fragment andlinker.

In some embodiments, the spacer unit that attaches the cleavable moietyto the eribulin drug moiety is self-immolative. In some embodiments, theself-immolative spacer unit comprises p-aminobenzyloxycarbonyl (pAB). Insome embodiments, the pAB spacer unit attaches the cleavable moiety tothe eribulin drug moiety via a C-35 amine. In some embodiments, thecleavable moiety is a cleavable peptide moiety, e.g., an amino acidunit. In some embodiments, the cleavable linker comprises Val-Cit-pAB.In some embodiments, the linker comprises Val-Cit-pAB and a PEG spacerunit joining the linker to the antibody moiety through a Mal moiety. Insome embodiments, the linker comprises Mal-(PEG)₂-Val-Cit-pAB.

In various embodiments, the antibody or antigen-binding fragment of theADC comprises human heavy and light chain variable region frameworks, orhuman heavy and light chain variable region frameworks with one or moreback mutations. In various embodiments, the antibody or antigen-bindingfragment of an ADC comprises a heavy chain variable region comprising anamino acid sequence of SEQ ID NO: 13 or that is at least 90% identicalto the amino acid sequence of SEQ ID NO 13, and a light chain variableregion comprising an amino acid sequence of SEQ ID NO: 14 or that is atleast 90% identical to the amino acid sequence of SEQ ID NO: 14. Invarious embodiments, the antibody or antigen-binding fragment of an ADCcomprises a human IgG1 heavy chain constant region comprising an aminoacid sequence of SEQ ID NO: 15, and a human Ig kappa light chainconstant region comprising an amino acid sequence of SEQ ID NO: 16. Invarious embodiments, the antibody or antigen-binding fragment of an ADCcomprises the heavy chain amino acid sequence of SEQ ID NO: 17, and thelight chain amino acid sequence of SEQ ID NO: 18.

In various embodiments, the ADC has Formula (I):Ab−(L−D)_(p)  (I)whereinAb is an antibody or antigen-binding fragment, wherein the antibody orantigen-binding fragment is capable of binding to mesothelin andcomprises three heavy chain complementarity determining regions (HCDRs)comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2(HCDR2), and SEQ ID NO: 3 (HCDR3); and three light chain complementaritydetermining regions (LCDRs) comprising amino acid sequences of SEQ IDNO: 4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as definedby the Kabat numbering system; or three heavy chain complementaritydetermining regions (HCDRs) comprising amino acid sequences of SEQ IDNO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3); and threelight chain complementarity determining regions (LCDRs) comprising aminoacid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQID NO: 12 (LCDR3), as defined by the IMGT numbering system;D is an eribulin;L is a cleavable linker comprising Mal-(PEG)₂-Val-Cit-pAB; andp is an integer from 1 to 8, e.g., p is an integer from 2 to 6 or 3 to4.

In some embodiments, p is an integer from 1 to 6. In some embodiments, pis 2 or 6.

In various embodiments, the antibody or antigen-binding fragment of theADC (e.g., the ADC described above) comprises human heavy and lightchain variable region frameworks, or human heavy and light chainvariable region frameworks with one or more back mutations. In variousembodiments, the antibody or antigen-binding fragment of the ADCcomprises a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 13 or that is at least 90% identical to SEQ IDNO: 13, and a light chain variable region comprising an amino acidsequence of SEQ ID NO: 14 or that is at least 90% identical to SEQ IDNO: 14. In various embodiments, the antibody or antigen-binding fragmentof the ADC comprises a human IgG1 heavy chain constant region comprisingan amino acid sequence of SEQ ID NO: 15, and a human Ig kappa lightchain constant region comprising an amino acid sequence of SEQ ID NO:16. In various embodiments, the antibody or antigen-binding fragment ofthe ADC comprises the heavy chain amino acid sequence of SEQ ID NO: 17,and the light chain amino acid sequence of SEQ ID NO: 18.

In various embodiments, the ADC has Formula I:Ab−(L−D)_(p)  (I)wherein Ab is an antibody or antigen-binding fragment, wherein theantibody or antigen-binding fragment is capable of binding to mesothelinand comprises a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 13, and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 14;D is eribulin;L is a cleavable linker comprising Mal-(PEG)₂-Val-Cit-pAB; andp is an integer from 1 to 8, e.g., p is an integer from 2 to 6 or 3 to4.

In some embodiments, p is an integer from 1 to 6. In some embodiments, pis 2 or 6.

In some embodiments, the antibody or antigen-binding fragment of the ADCcomprises a human IgG1 heavy chain constant region, and a human Ig kappalight chain constant region. In some embodiments, the antibody orantigen-binding fragment comprises an IgG1 heavy chain constant regioncomprising an amino acid sequence of SEQ ID NO: 15, and an Ig kappalight chain constant region comprising an amino acid sequence of SEQ IDNO: 16. In some embodiments, the antibody or antigen-binding fragmentcomprises a heavy chain comprising an amino acid sequence of SEQ ID NO:17, and a light chain comprising an amino acid sequence of SEQ ID NO:18.

In various embodiments, provided herein are pharmaceutical compositionscomprising the described antibodies, antigen-binding fragments,conjugates, and/or ADC compositions. In some embodiments, thepharmaceutical composition comprises one or more antibodies orantigen-binding fragments and/or one or more ADCs described herein alongwith at least a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical composition comprises multiple copies ofthe antibody, antigen-binding fragment, and/or ADC. In some embodiments,the pharmaceutical composition comprises multiple copies of an ADCdisclosed herein, wherein the average p of the ADC is about 1 to about6. In some embodiments, the average p of the ADC in the composition isabout 1.7 or 2, or about 6.

In various embodiments, provided herein are therapeutic uses for thedescribed antibodies, antigen-binding fragments, conjugates, and/or ADCcompositions, e.g., in treating cancer. In certain aspects, the presentdisclosure provides methods of treating a cancer that expresses anantigen targeted by the antibody, antigen-binding fragment, and/or theantibody moiety of the conjugate or ADC, such as mesothelin. In certainaspects, the present disclosure provides methods of killing orinhibiting the proliferation of tumor cells or cancer cells byadministering a therapeutically effective amount and/or regimen of anyone of the antibodies, antigen-binding fragments, conjugates, and/orADCs described herein. In some embodiments, the cancer is amesothelin-expressing cancer, such as a mesothelioma, a breast cancer, acervical cancer, a colorectal cancer, an endometrial cancer, a head andneck cancer, a liver cancer, a lung cancer (e.g., a non-small cell lungcancer), an ovarian cancer (e.g., a serous or a clear cell ovariancancer), a pancreatic cancer, a prostate cancer, a renal cancer, agastric cancer, a thyroid cancer, a urothelial cancer, a uterine cancer,a bile duct cancer, or a leukemia.

In certain aspects, the present disclosure provides uses for thedescribed antibodies, antigen-binding fragments, conjugates, and/or ADCcompounds and compositions, e.g., for determining whether a subjecthaving or suspected of having a cancer (e.g., a mesothelin-expressingcancer) will be responsive to treatment with an agent targetingmesothelin, e.g., an antibody or antibody binding fragment, conjugate,or ADC disclosed herein. In some embodiments, the method comprisesproviding a biological sample from the subject; contacting the samplewith an antibody or antigen-binding fragment disclosed herein; anddetecting binding of the antibody or antigen-binding fragment to one ormore cancer cells in the sample.

In certain other aspects, the present disclosure provides pharmaceuticalcompositions comprising an antibody or antibody binding fragment,conjugate, and/or ADC and a pharmaceutically acceptable diluent,carrier, and/or excipient. Methods of producing the disclosed antibodyor antibody binding fragment, conjugate, or ADC compounds andcompositions are also provided.

In some embodiments, nucleic acid sequence(s) encoding an antibody orantigen-binding fragment, a conjugate, or an ADC of the presentdisclosure are provided. The nucleic acid(s) may be in the form of anisolated nucleic acid, a nucleic acid incorporated into an isolatedvector comprising, and/or an antibody or antigen-binding fragmentexpressed by a cell population under conditions suitable to produce theantibody or antigen-binding fragment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows detection of the specific reactivity of immune sera againsthuman mesothelin by flow cytometry.

FIG. 2 shows detection of the specific reactivity of culturesupernatants to human mesothelin by ELISA.

FIG. 3 shows In-Fusion PCR amplification of anti-mesothelin antibodiesby gel electrophoresis.

FIG. 4 shows anti-mesothelin clones for In-Fusion cloning andexpression.

FIG. 5 shows a summary of purified 48 Rb-hu-xi anti-mesothelinantibodies.

FIG. 6 shows in-vitro cell-based potency results for anti-mesothelin-AuFconjugates.

FIG. 7 shows epitope binning characterization of anti-mesothelinantibodies.

FIG. 8 shows DSC analysis results for humanized 345A12 antibodies.345A12 F(ab′)2 fragments were subject to thermal analysis ranging from25-100° C. using a scan rate of 100° C./hour.

FIG. 9 shows stability of MORAb-109 (345A12-HC15-LC4-VCP-eribulin)(DAR2) in various matrices.

FIG. 10A and FIG. 10B show the anti-tumor effect (FIG. 10A) and bodyweight change (FIG. 10B) in a human non-small cell lung cancer(NSCLC)NCI-H2110 xenograft model treated with 345A12-HC1-LC2-diOHeribulin dimer ADC at 2.5 mg/kg or 102A6A2-HC1-LC2-diOH eribulin dimerADC at 2.5 mg/kg (Study M109-004-2016).

FIG. 11A and FIG. 11B show body weight change of female CD-1 micetreated with 345A12-HC1-LC2-diOH eribulin dimer ADC (5, 10, 15, or 20mg/kg) (FIG. 11A) or 345A12-HC15-LC4-diOH eribulin dimer ADC (5, 10, or20 mg/kg) (FIG. 11B) (Study M109-006-2017).

FIG. 12A and FIG. 12B show the anti-tumor effect (FIG. 12A) and bodyweight change (FIG. 12B) in a human NSCLC NCI-H2110 xenograft modeltreated with 345A12-HC10-LC4-diOH eribulin dimer ADC at 2.5 mg/kg or345A12-HC15-LC4-diOH eribulin dimer ADC at 2.5 mg/kg (StudyM109-007-2017).

FIG. 13A and FIG. 13B show the anti-tumor effect (FIG. 13A) and bodyweight change (FIG. 13B) in a human gastric cancer NCI-N87 xenograftmodel treated with MORAb-109 (DAR2 or DAR6) (Study M109-010-2018).

FIG. 14A and FIG. 14B show the anti-tumor effect (FIG. 14A) and bodyweight change (FIG. 14B) in a human mesothelioma HAY xenograft modeltreated with MORAb-109 (DAR2 or DAR6) or eribulin (Study M109-010-2018).

FIG. 15A and FIG. 15B show the anti-tumor effect (FIG. 15A) and bodyweight change (FIG. 15B) in a human mesothelioma PDX model (Meso7212)treated with MORAb-109 (DAR6) or eribulin.

FIG. 16A and FIG. 16B show the anti-tumor effect (FIG. 16A) and bodyweight change (FIG. 16B) in a human mesothelioma PDX model (Meso7212)treated with different DAR species of MORAb-109 or eribulin.

FIG. 17 shows a correlation analysis between mesothelin (MSLN)expression and in vitro potency (IC₅₀) of eribulin and MORAb-109 (DAR2and DAR6) in various cell lines. The correlation for MORAb-109 (DAR2)was analyzed in all 51 cell lines and in a subset of cell lines withhigher mesothelin expression levels (FACS staining of mean fluorescenceintensity (MFI) equal to or >80). The subset excluded cell lines withlower mesothelin expression levels (FACS staining of MFI<80).

FIG. 18A-C show the anti-tumor effect (FIG. 18A and FIG. 18B) and bodyweight change (FIG. 18C) in a human gastric cancer NCI-N87 xenograftmodel treated with different doses of MORAb-109 (DAR2) ranging from 5mg/kg to 25 mg/kg.

FIG. 19 shows the concentrations (μg/mL) of total and intact MORAb-109(DAR2) in NCI-N87 tumor-bearing mice following treatment with differentdoses of MORAb-109 (DAR2) ranging from 5 mg/kg to 25 mg/kg.

FIG. 20A and FIG. 20B show the anti-tumor effect (FIG. 20A) and bodyweight change (FIG. 20B) in a human ovarian cancer OVCAR-3-A1-T1xenograft model treated with MORAb-109 (DAR2) (5 mg/kg) or eribulin (0.1or 3.2 mg/kg).

FIG. 21A and FIG. 21B show the anti-tumor effect (FIG. 21A) and bodyweight change (FIG. 21B) in a human NSCLC PDX model (LC-F-25) treatedwith MORAb-109 (DAR2) (10 mg/kg) or eribulin (0.1 or 3.2 mg/kg).

FIG. 22A and FIG. 22B show the anti-tumor effect (FIG. 22A) and bodyweight change (FIG. 22B) in a human NSCLC PDX model (LXFA-737) treatedwith MORAb-109 (DAR2) (10 mg/kg) or eribulin (0.2 or 3.2 mg/kg).

FIG. 23A and FIG. 23B show the anti-tumor effect (FIG. 23A) and bodyweight change (FIG. 23B) in a human gastric cancer NCI-N87 xenograftmodel treated with a single dose of MORAb-109 (DAR2 or DAR6) at 10 mg/kg(3 mice per group).

FIG. 24A and FIG. 24B show the DAR of MORAb-109 (DAR2) (FIG. 24A) orMORAb-109 (DAR6) (FIG. 24B) in plasma samples from NCI-N87 tumor-bearingmice after treatment with a single dose of MORAb-109 (DAR2 or DAR6) at10 mg/kg.

FIG. 25A and FIG. 25B show the cytotoxicity (% killing) of MORAb-109(DAR2) (FIG. 25A) or BAY 94-9343 (FIG. 25B) on NCI-N87 gastric cancercells. Both anti-MSLN ADCs were evaluated alone and in the presence ofunconjugated antibody.

FIG. 26A and FIG. 26B show the ADCC activity of MORAb-109 (DAR2) and345A12-HC15-LC4 (FIG. 26A) or BAY 94-9343 and anetumab (FIG. 26B), asmeasured by a luciferase assay. ADCC activity was calculated by relativearea under the curve (AUC).

FIG. 27 shows a stability analysis of anti-MSLN ADCs, MORAb-109 (DAR2)and BAY 94-9343, in mouse and human plasma.

FIG. 28A and FIG. 28B show the anti-tumor effect (FIG. 28A) and bodyweight change (FIG. 28B) in a human gastric cancer NCI-N87 xenograftmodel treated with MORAb-109 (DAR2) (5 mg/kg), BAY 94-9343 (5 mg/kg), oreribulin (1 mg/kg).

FIG. 29A and FIG. 29B show the anti-tumor effect (FIG. 29A) and bodyweight change (FIG. 29B) in a human mesothelioma HAY xenograft modeltreated with MORAb-109 (DAR2) (5 mg/kg), BAY 94-9343 (5 mg/kg), oreribulin (1 mg/kg).

FIG. 30A and FIG. 30B show the anti-tumor effect (FIG. 30A) and bodyweight change (FIG. 30B) in a human mesothelioma PDX model (Meso7212)treated with MORAb-109 (DAR2) (10 mg/kg), BAY 94-9343 (10 mg/kg),eribulin (1 mg/kg), or DM4 (0.3 mg/kg).

FIG. 31A and FIG. 31B show the anti-tumor effect (FIG. 31A) and bodyweight change (FIG. 31B) in a human NSCLC PDX model (LXFA-586) treatedwith MORAb-109 (DAR2) (25 mg/kg), BAY 94-9343 (DAR˜4) (25 mg/kg), oreribulin (3.2 mg/kg).

FIG. 32A and FIG. 32B show the anti-tumor effect (FIG. 32A) and bodyweight change (FIG. 32B) in a human NSCLC PDX model (LXFL-529) treatedwith MORAb-109 (DAR2) (25 mg/kg), MORAb-109 (DAR2) (12.5 mg/kg),MORAb-109 (DAR2) (12.5 mg/kg, QW×3), BAY 94-9343 (DAR˜4) (12.5 mg/kg),or eribulin (3.2 mg/kg).

DETAILED DESCRIPTION

The disclosed compositions and methods may be understood more readily byreference to the following detailed description taken in connection withthe accompanying figures, which form a part of this disclosure. It is tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed compositions and methods unlessthe context indicates otherwise.

Throughout this text, the descriptions refer to compositions and methodsof using the compositions. Where the disclosure describes or claims afeature or embodiment associated with a composition, such a feature orembodiment is equally applicable to the methods of using thecomposition. Likewise, where the disclosure describes or claims afeature or embodiment associated with a method of using a composition,such a feature or embodiment is equally applicable to the composition.

When a range of values is expressed, it includes embodiments using anyparticular value within the range. Further, reference to values statedin ranges includes each and every value within that range. All rangesare inclusive of their endpoints and combinable. When values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment.Reference to a particular numerical value includes at least thatparticular value, unless the context clearly dictates otherwise. The useof “or” will mean “and/or” unless the specific context of its usedictates otherwise. All references cited herein are incorporated byreference for any purpose. Where a reference and the specificationconflict, the specification will control.

It is to be appreciated that certain features of the disclosedcompositions and methods, which are, for clarity, described herein inthe context of separate embodiments, may also be provided in combinationin a single embodiment. Conversely, various features of the disclosedcompositions and methods that are, for brevity, described in the contextof a single embodiment, may also be provided separately or in anysubcombination.

Definitions

Various terms relating to aspects of the description are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinitions provided herein.

As used herein, the singular forms “a,” “an,” and “the” include pluralforms unless the context clearly dictates otherwise.

The terms “about” or “approximately” in the context of numerical valuesand ranges refers to values or ranges that approximate or are close tothe recited values or ranges such that the embodiment may perform asintended, such as having a desired amount of nucleic acids orpolypeptides in a reaction mixture, as is apparent to the skilled personfrom the teachings contained herein. Thus, these terms encompass valuesbeyond those resulting from systematic error. In some embodiments, aboutmeans plus or minus 10% of a numerical amount.

The terms “antibody-drug conjugate,” “antibody conjugate,” “conjugate,”“immunoconjugate,” and “ADC” are used interchangeably, and refer to atherapeutic compound (e.g., an eribulin moiety) that is linked to anantibody moiety and is defined by the generic formula: Ab-(L-D)_(p)(Formula I), wherein Ab is an antibody moiety (e.g., an antibody orantigen-binding fragment), L is a linker moiety, D is a drug moiety(e.g., an eribulin drug moiety), and p is the number of drug moietiesper antibody moiety. In ADCs comprising an eribulin drug moiety, “p”refers to the number of eribulin moieties linked to the antibody moiety.In some embodiments, the linker L can include a cleavable moiety thatcan either directly attach to the antibody moiety and to the therapeuticcompound, or the cleavable moiety can be attached to either or both theantibody moiety and therapeutic compound by spacer unit(s). In someembodiments, when a spacer unit attaches the cleavable moiety to thetherapeutic compound, it is a self-immolative spacer unit.

The term “antibody” is used in the broadest sense to refer to animmunoglobulin molecule that recognizes and specifically binds to atarget, such as a protein, polypeptide, carbohydrate, polynucleotide,lipid, or combinations of the foregoing through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. The heavy chain of an antibody is composed of a heavy chainvariable domain (VH) and a heavy chain constant region (CH). The lightchain is composed of a light chain variable domain (VL) and a lightchain constant domain (CL). For the purposes of this application, themature heavy chain and light chain variable domains each comprise threecomplementarity determining regions (CDR1, CDR2, and CDR3) within fourframework regions (FR1, FR2, FR3, and FR4) arranged from N-terminus toC-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An “antibody” canbe naturally occurring or man-made, such as monoclonal antibodiesproduced by conventional hybridoma technology. The term “antibody”includes full-length monoclonal antibodies and full-length polyclonalantibodies, as well as antibody fragments such as Fab, Fab′, F(ab′)2,Fv, and single chain antibodies. An antibody can be any one of the fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, orsubclasses thereof (e.g., isotypes IgG1, IgG2, IgG3, IgG4). The termfurther encompasses human antibodies, chimeric antibodies, humanizedantibodies and any modified immunoglobulin molecule containing anantigen recognition site, so long as it demonstrates the desiredbiological activity.

The term “monoclonal antibody,” as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present disclosure may be made by the hybridomamethod first described by Kohler et al. (1975) Nature 256:495, or may bemade by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).Monoclonal antibodies may also be isolated from phage antibody librariesusing the techniques described in Clackson et al. (1991) Nature352:624-8, and Marks et al. (1991) J. Mol. Biol. 222:581-97, forexample.

The monoclonal antibodies described herein specifically include“chimeric” antibodies, in which a portion of the heavy and/or lightchain is identical with or homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey specifically bind the target antigen and/or exhibit the desiredbiological activity.

The term “chimeric antibody,” as used herein, refers to antibodieswherein the amino acid sequence of the immunoglobulin molecule isderived from two or more species. In some instances, the variableregions of both heavy and light chains corresponds to the variableregions of antibodies derived from one species with the desiredspecificity, affinity, and activity while the constant regions arehomologous to antibodies derived from another species (e.g., human) tominimize an immune response in the latter species.

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., rabbit)antibodies as well as human antibodies. Such antibodies are chimericantibodies which contain minimal sequences derived from non-humanimmunoglobulin. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions are those of a human immunoglobulin sequence.The humanized antibody optionally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. The humanized antibody can be further modified by thesubstitution of residues, either in the Fv framework region and/orwithin the replaced non-human residues to refine and optimize antibodyspecificity, affinity, and/or activity.

The term “antigen-binding fragment,” “antigen-binding domain,” or“antigen-binding portion” of an antibody, as used herein, refers to oneor more fragments of an antibody or protein that retain the ability tospecifically bind to an antigen (e.g., mesothelin). Antigen-bindingfragments may also retain the ability to internalize into anantigen-expressing cell. In some embodiments, antigen-binding fragmentsalso retain immune effector activity. It has been shown that fragmentsof a full-length antibody can perform the antigen-binding function of afull-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding fragment,” “antigen-binding domain,” or“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii)a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody; (v) a dAbfragment, which comprises a single variable domain, e.g., a VH domain(see, e.g., Ward et al. (1989) Nature 341:544-6; and Intl. Pub. No. WO1990/005144); and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv)). See, e.g., Birdet al. (1988) Science 242:423-6; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-83. Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding fragment” or“antigen-binding portion” of an antibody, and are known in the art as anexemplary type of binding fragment that can internalize into cells uponbinding (see, e.g., Zhu et al. (2010) 9:2131-41; He et al. (2010) J.Nucl. Med. 51:427-32; and Fitting et al. (2015) MAbs 7:390-402). Incertain embodiments, scFv molecules may be incorporated into a fusionprotein. Other forms of single chain antibodies, such as diabodies arealso encompassed. Diabodies are bivalent, bispecific antibodies in whichVH and VL domains are expressed on a single polypeptide chain, but usinga linker that is too short to allow for pairing between the two domainson the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen-bindingsites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-8; and Poljak et al. (1994) Structure 2:1121-3). Antigen-bindingfragments are obtained using conventional techniques known to those ofskill in the art, and the binding fragments are screened for utility(e.g., binding affinity, internalization) in the same manner as areintact antibodies. Antigen-binding fragments may be prepared by cleavageof the intact protein, e.g., by protease or chemical cleavage.

“Internalizing” as used herein in reference to an antibody orantigen-binding fragment refers to an antibody or antigen-bindingfragment that is capable of being taken through the cell's lipid bilayermembrane to an internal compartment (i.e., “internalized”) upon bindingto the cell, typically into a degradative compartment in the cell. Forexample, an internalizing anti-mesothelin antibody is one that iscapable of being taken into the cell after binding to mesothelin on thecell membrane. In some embodiments, the antibody or antigen-bindingfragment used in the ADCs disclosed herein targets a cell surfaceantigen (e.g., mesothelin) via an internalizing antibody orinternalizing antigen-binding fragment (allowing the ADC to transferthrough the cellular membrane after antigen-binding).

The term “mesothelin” or “MSLN,” as used herein, refers to any nativeform of human mesothelin (MSLN). The term encompasses full-lengthmesothelin (e.g., NCBI Reference Sequence: AAC50348.1), as well as anyform of human mesothelin that results from cellular processing. The termalso encompasses naturally occurring variants of mesothelin, includingbut not limited to splice variants, allelic variants, and isoforms.Mesothelin can be isolated from a human, or may be producedrecombinantly or by synthetic methods. The term may also encompass anysynthetic variant to which an anti-mesothelin antibody, e.g., anantibody disclosed herein, and/or antigen-binding fragment, canspecifically bind.

The term “anti-mesothelin antibody” or “antibody that specifically bindsmesothelin” refers to any form of antibody or fragment thereof thatspecifically binds mesothelin, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as theyspecifically bind mesothelin. In some embodiments, the anti-mesothelinantibody used in the ADCs disclosed herein is an internalizing antibodyor internalizing antibody fragment. 345A12 (e.g., 345A12-HC15-LC4) and102A6A2 are exemplary internalizing anti-human mesothelin antibodies. Asused herein, the terms “specific,” “specifically binds,” and “bindsspecifically” refer to the selective binding of the antibody to thetarget antigen epitope. Antibodies can be tested for specificity ofbinding by comparing binding to an appropriate antigen to binding to anirrelevant antigen or antigen mixture under a given set of conditions.If the antibody binds to the appropriate antigen with at least 2, 5, 7,or 10 times more affinity than to the irrelevant antigen or antigenmixture, then it is considered to be specific. A “specific antibody” or“target-specific antibody” is one that only binds the target antigen(e.g., mesothelin), but does not bind (or exhibits minimal binding) toother antigens.

The term “epitope” refers to the portion of an antigen capable of beingrecognized and specifically bound by an antibody. When the antigen is apolypeptide, epitopes can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of thepolypeptide. The epitope bound by an antibody may be identified usingany epitope mapping technique known in the art, including X-raycrystallography for epitope identification by direct visualization ofthe antigen-antibody complex, as well as monitoring the binding of theantibody to fragments or mutated variations of the antigen, ormonitoring solvent accessibility of different parts of the antibody andthe antigen. Exemplary strategies used to map antibody epitopes include,but are not limited to, array-based oligo-peptide scanning, limitedproteolysis, site-directed mutagenesis, high-throughput mutagenesismapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g.,Gershoni et al. (2007) 21:145-56; and Hager-Braun and Tomer (2005)Expert Rev. Proteomics 2:745-56).

Competitive binding and epitope binning can also be used to determineantibodies sharing identical or overlapping epitopes. Competitivebinding can be evaluated using a cross-blocking assay, such as the assaydescribed in “Antibodies, A Laboratory Manual,” Cold Spring HarborLaboratory, Harlow and Lane (1^(st) edition 1988, 2^(nd) edition 2014).In some embodiments, competitive binding is identified when a testantibody or binding protein reduces binding of a reference antibody orbinding protein to a target antigen such as mesothelin (e.g., a bindingprotein comprising CDRs and/or variable domains selected from thoseidentified in Tables 1-3), by at least about 50% in the cross-blockingassay (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or anypercentage in between), and/or vice versa. In some embodiments,competitive binding can be due to shared or similar (e.g., partiallyoverlapping) epitopes, or due to steric hindrance where antibodies orbinding proteins bind at nearby epitopes (see, e.g., Tzartos, Methods inMolecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66)). In someembodiments, competitive binding can be used to sort groups of bindingproteins that share similar epitopes. For example, binding proteins thatcompete for binding can be “binned” as a group of binding proteins thathave overlapping or nearby epitopes, while those that do not compete areplaced in a separate group of binding proteins that do not haveoverlapping or nearby epitopes.

The term “k_(on)” or “k_(a)” refers to the on-rate constant forassociation of an antibody to the antigen to form the antibody/antigencomplex. The rate can be determined using standard assays, such as asurface plasmon resonance, biolayer inferometry, or ELISA assay.

The term “k_(off)” or “k_(a)” refers to the off-rate constant fordissociation of an antibody from the antibody/antigen complex. The ratecan be determined using standard assays, such as a surface plasmonresonance, biolayer inferometry, or ELISA assay.

The term “K_(D)” refers to the equilibrium dissociation constant of aparticular antibody-antigen interaction. K_(D) is calculated byk_(a)/k_(a). The rate can be determined using standard assays, such as asurface plasmon resonance, biolayer inferometry, or ELISA assay.

The term “p” or “drug loading” or “drug:antibody ratio” or“drug-to-antibody ratio” or “DAR” refers to the number of drug moietiesper antibody moiety, i.e., drug loading, or the number of -L-D moietiesper antibody or antigen-binding fragment (Ab) in ADCs of Formula (I). InADCs comprising an eribulin drug moiety, “p” refers to the number oferibulin moieties linked to the antibody moiety. For example, if twoeribulin moieties are linked to an antibody moiety, p=2. In compositionscomprising multiple copies of ADCs of Formula (I), “average p” refers tothe average number of -L-D moieties per antibody or antigen-bindingfragment in a population of ADCs, also referred to as “average drugloading.”

A “linker” or “linker moiety” is used herein to refer to any chemicalmoiety that is capable of covalently joining a compound, usually a drugmoiety such as eribulin, to another moiety such as an antibody moiety.Linkers can be susceptible to or substantially resistant to acid-inducedcleavage, peptidase-induced cleavage, light-based cleavage,esterase-induced cleavage, and/or disulfide bond cleavage, at conditionsunder which the compound or the antibody remains active.

The term “agent” is used herein to refer to a chemical compound, amixture of chemical compounds, a biological macromolecule, or an extractmade from biological materials. The term “therapeutic agent” or “drug”refers to an agent that is capable of modulating a biological processand/or has biological activity. The eribulin monomer described herein isan exemplary therapeutic agent.

The term “chemotherapeutic agent” or “anti-cancer agent” is used hereinto refer to all agents that are effective in treating cancer regardlessof mechanism of action. Inhibition of metastasis or angiogenesis isfrequently a property of a chemotherapeutic agent. Chemotherapeuticagents include antibodies, biological molecules, and small molecules,and encompass eribulin, as described herein. A chemotherapeutic agentmay be a cytotoxic or cytostatic agent. The term “cytostatic agent”refers to an agent that inhibits or suppresses cell growth and/ormultiplication of cells. The term “cytotoxic agent” refers to asubstance that causes cell death primarily by interfering with a cell'sexpression activity and/or functioning.

The term “eribulin” or “eribulin monomer,” as used herein, refers to asynthetic analog of halichondrin B, a macrocyclic compound that wasoriginally isolated from the marine sponge Halichondria okadais.Eribulin is a microtubule dynamics inhibitor, which is thought to bindtubulin and induce cell cycle arrest at the G2/M phase by inhibitingmitotic spindle assembly. The term “eribulin mesylate” refers to themesylate salt of eribulin, which is marketed under the trade nameHalaven™. Exemplary eribulin analogs include those shown and describedin U.S. Pat. Nos. 6,214,865 and 6,653,341, which are incorporated hereinby reference for the disclosed eribulin structures and methods ofsynthesizing those structures.

The term “eribulin dimer,” as used herein, refers to a dimeric form oferibulin in which two eribulin monomers are attached via a covalent ornon-covalent bond either directly or by a chemical linker (e.g., asecondary amine, a dihydroxyl secondary amine). Eribulin dimers may, insome embodiments, consist of two eribulin monomers covalently linked atthe C-34 position by a secondary amine, or two eribulin monomerscovalently linked at the C-35 position by a dihydroxyl secondary amine.An eribulin dimer consisting of two eribulin monomers covalently linkedat the C-34 position by a secondary amine may be referred to herein as a“desOH eribulin dimer.” An eribulin dimer consisting of two eribulinmonomers covalently linked at the C-35 position by a dihydroxylsecondary amine may be referred to herein as a “diOH eribulin dimer.”The term “eribulin dimer drug moiety” refers to the component of an ADCor composition that provides the structure of an eribulin dimer, e.g.,the eribulin dimer (D) component in an ADC of Formula (I), or in acomposition comprising -L-D. In some embodiments, a desOH eribulin dimerand/or a diOH eribulin dimer provides improved conjugatability overother eribulin dimer formats.

The term “cryptophycin,” as used herein, refers to cryptophycin-1, amacrolide compound that was originally isolated from the cyanobacteriumNostoc, or to any synthetic analog thereof retaining anti-tubulinactivity. Exemplary cryptophycin analogs include those shown anddescribed in Intl. Publ. No. WO 2017/136769, which is incorporatedherein by reference for all its disclosed cryptophycin structures andmethods of synthesizing those structures. The term “cryptophycin drugmoiety” refers to the component of an ADC or composition that has thestructure of a cryptophycin.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

The term “inhibit” or “inhibition of,” as used herein, means to reduceby a measurable amount, and can include but does not require completeprevention or inhibition.

The term “bystander killing” or “bystander effect” refers to the killingof target-negative cells in the presence of target-positive cells,wherein killing of target-negative cells is not observed in the absenceof target-positive cells. Cell-to-cell contact, or at least proximitybetween target-positive and target-negative cells, enables bystanderkilling. This type of killing is distinguishable from “off-targetkilling,” which refers to the indiscriminate killing of target-negativecells. “Off-target killing” may be observed in the absence oftarget-positive cells.

The term “cancer” refers to the physiological condition in mammals inwhich a population of cells is characterized by unregulated cell growth.Examples of cancers include, but are not limited to, a carcinoma,lymphoma, blastoma, sarcoma, and leukemia. Particular examples of suchcancers include mesothelin-expressing cancers, such as a mesothelioma, abreast cancer, a cervical cancer, a colorectal cancer, an endometrialcancer, a head and neck cancer, a liver cancer, a lung cancer (e.g., anon-small cell lung cancer), an ovarian cancer (e.g., a serous, a clearcell, or an epithelial ovarian cancer), a pancreatic cancer, a prostatecancer, a renal cancer, a gastric cancer, a thyroid cancer, a urothelialcancer, a uterine cancer, a bile duct cancer, or a leukemia.

The terms “tumor” and “neoplasm” refer to any mass of tissue thatresults from excessive cell growth or proliferation, either benign ormalignant, including precancerous lesions.

The terms “tumor cell” refer to individual cells or the total populationof cells derived from a tumor, including both non-tumorigenic cells andcancer stem cells. As used herein, the term “tumor cell” will bemodified by the term “non-tumorigenic” when referring solely to thosetumor cells lacking the capacity to renew and differentiate todistinguish those tumor cells from cancer stem cells.

The terms “subject” and “patient” are used interchangeably herein torefer to any animal, such as any mammal, including but not limited to,humans, non-human primates, rodents, and the like. In some embodiments,the mammal is a mouse. In some embodiments, the mammal is a human.

A “pharmaceutical composition” refers to a preparation which is in suchform as to permit administration and subsequently provide the intendedbiological activity of the active ingredient(s) and/or to achieve atherapeutic effect, and which contains no additional components whichare unacceptably toxic to a subject to which the formulation would beadministered. The pharmaceutical composition may be sterile.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government, or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia, for use inanimals, and more particularly in humans.

An “effective amount” of, e.g., an antibody, antigen-binding fragment,and/or ADC as disclosed herein is an amount sufficient to perform aspecifically stated purpose, for example to produce a therapeutic effectafter administration, such as a reduction in tumor growth rate or tumorvolume, a reduction in a symptom of cancer, or some other indicia oftreatment efficacy. An effective amount can be determined in a routinemanner in relation to the stated purpose. The term “therapeuticallyeffective amount” refers to an amount of an antibody, antigen-bindingfragment, and/or an ADC effective to treat a disease or disorder in asubject. In the case of cancer, a therapeutically effective amount of anantibody, antigen-binding fragment, and/or ADC can reduce the number ofcancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumormetastasis, inhibit (e.g., slow or stop) tumor growth, and/or relieveone or more symptoms. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. Typically, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

As used herein, “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality. As isreadily appreciated in the art, full eradication of disease isencompassed but not required for a treatment act. “Treatment” or“treat,” as used herein, refers to the administration of a describedantibody, antigen-binding fragment, and/or ADC to a subject, e.g., apatient. The treatment can be to cure, heal, alleviate, relieve, alter,remedy, ameliorate, palliate, improve or affect the disorder, thesymptoms of the disorder or the predisposition toward the disorder,e.g., a cancer. In some embodiments, in addition to treating a subjectwith a condition, a composition disclosed herein can also be providedprophylactically to prevent or reduce the likelihood of developing thatcondition.

In some embodiments, a labeled antibody, antigen-binding fragment,and/or ADC is used. Suitable “labels” include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent moieties,chemiluminescent moieties, magnetic particles, and the like.

By “protein,” as used herein, is meant at least two covalently attachedamino acids. The term encompasses polypeptides, oligopeptides, andpeptides. In some embodiments, the two or more covalently attached aminoacids are attached by a peptide bond. The protein may be made up ofnaturally occurring amino acids and peptide bonds, for example when theprotein is made recombinantly using expression systems and host cells.Alternatively, the protein may include synthetic amino acids (e.g.,homophenylalanine, citrulline, ornithine, and norleucine). A“recombinant protein” is a protein made using recombinant techniquesusing any techniques and methods known in the art, i.e., through theexpression of a recombinant nucleic acid. Methods and techniques for theproduction of recombinant proteins are well known in the art.

For amino acid sequences, sequence identity and/or similarity may bedetermined using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman (1981) Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman (1988) Proc. Nat.Acad. Sci. USA 85:2444, computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.), theBest Fit sequence program described by Devereux et al. (1984) Nucl. AcidRes. 12:387-95, e.g., using the default settings, or by inspection. Insome embodiments, percent identity is calculated by FastDB based uponthe following parameters: mismatch penalty of 1; gap penalty of 1; gapsize penalty of 0.33; and joining penalty of 30 (“Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp. 127-149 (1988), AlanR. Liss, Inc).

Generally, the amino acid homology, similarity, or identity betweenproteins disclosed herein and variants thereof, including variants oftarget antigens (such as mesothelin), variants of tubulin sequences, andvariants of antibody variable domains (including individual variantCDRs), are at least 80% to the sequences depicted herein, e.g.,homologies or identities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, almost 100%, or 100%.

In a similar manner, “percent (%) nucleic acid sequence identity” withrespect to the nucleic acid sequence of the antibodies and otherproteins identified herein is defined as the percentage of nucleotideresidues in a candidate sequence that are identical with the nucleotideresidues in the coding sequence of the antigen-binding protein. Aspecific method utilizes the BLASTN module of WU-BLAST-2 set to thedefault parameters, with overlap span and overlap fraction set to 1 and0.125, respectively.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed antigen-binding protein CDRvariants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, for example, MI3 primermutagenesis and PCR mutagenesis.

Anti-Mesothelin Antibodies and Antigen-Binding Fragments

The present disclosure relates, in various embodiments, to antibodies orantigen-binding fragments thereof capable of binding and/or killingtumor cells (e.g., mesothelin-expressing tumor cells), as well as theiruse in conjugates and therapeutic compositions.

In some embodiments, the antibodies may be used alone, administered aspart of pharmaceutical compositions or combination therapies, and/or asthe antibody moiety in an ADC. In some embodiments, the anti-mesothelinantibodies and antigen-binding fragments disclosed herein are useful ontheir own (i.e., in unconjugated form) and as the antibody moiety in anADC. In some embodiments, the anti-mesothelin antibodies andantigen-binding fragments are humanized. In some embodiments, theanti-mesothelin antibodies and antigen-binding fragments contain minimalsequence derived from a non-human immunoglobulin and retain thereactivity of a non-human (e.g., rabbit) antibody while being lessimmunogenic in human. In some embodiments, the anti-mesothelinantibodies and antigen-binding fragments disclosed herein provide one ormore of improved stability, formulatability, aggregation, bindingaffinity, therapeutic efficacy, off-target toxicity, and/or metabolicprofile as compared to one or more anti-mesothelin antibodies known tothose of skill in the art.

In various embodiments, the antibodies or antigen-binding fragmentsdisclosed herein bind specifically to mesothelin, e.g., as expressed ona cancer cell. The antibody or antigen-binding fragment may bind to atarget antigen with a dissociation constant (K_(D)) of ≤1 mM, ≤100 nM or≤10 nM, or any amount in between, as measured by, e.g., BIAcore®analysis. In certain embodiments, the K_(D) is 1 pM to 500 pM. In someembodiments, the K_(D) is between 500 pM to 1 μM, 1 μM to 100 nM, or 100mM to 10 nM.

In some embodiments, the antibody moiety is a four-chain antibody (alsoreferred to as an immunoglobulin), comprising two heavy chains and twolight chains. In some embodiments the antibody moiety is a two-chainhalf body (one light chain and one heavy chain), or an antigen-bindingfragment of an immunoglobulin.

In some embodiments, the antibody moiety is an antibody orantigen-binding fragment thereof. In some embodiments, the antibodymoiety is an internalizing antibody or internalizing antigen-bindingfragment thereof. In some embodiments, the internalizing antibody orinternalizing antigen-binding fragment thereof binds to a target cancerantigen expressed on the surface of a cell and enters the cell uponbinding. In some embodiments, the eribulin drug moiety of the ADC isreleased from the antibody moiety of the ADC after the ADC enters and ispresent in a cell expressing the target cancer antigen (i.e., after theADC has been internalized).

In various embodiments, the antibody or antigen-binding fragmentdisclosed herein may comprise a paired set of heavy and light chainvariable domains taken from those listed in Tables 3-5, or the set ofsix CDR sequences from the paired heavy and light chain set, e.g., a setof CDRs listed in Tables 1-2. In some embodiments, the antibody orantigen-binding fragment further comprises human heavy and light chainframeworks (optionally with one or more backmutations to improve bindingaffinity) and/or human heavy and light chain constant domains orfragments thereof. For instance, the antibody or antigen-bindingfragment may comprise a human IgG heavy chain constant domain (such asan IgG1) and a human kappa or lambda light chain constant domain. Insome embodiments, the antibody or antigen-binding fragment comprises ahuman immunoglobulin G subtype 1 (IgG1) heavy chain constant domain witha human Ig kappa light chain constant domain.

Amino acid and nucleic acid sequences of exemplary antibodies of thepresent disclosure are set forth in Tables 1-10.

TABLE 1 Amino acid sequences of Kabat CDRs for ananti-mesothelin antibody SEQ mAb IgG chain ID Amino acid sequence345A12- HC CDR1 1 SYAMS HC15-LC4 HC CDR2 2 VIDISGNRFYADWVKG HC CDR3 3VDSRAWGPFNL LC CDR1 4 QASQSIFSYLA LC CDR2 5 DASDLAS LC CDR3 6QQGYTRSDVDNA

TABLE 2 Amino acid sequences of IMGT CDRs for ananti-mesothelin antibody SEQ mAb IgG chain ID Amino acid sequence345A12- HC CDR1  7 GIDLSSYA HC15-LC4 HC CDR2  8 IDISGNR HC CDR3  9ARVDSRAWGPFNL LC CDR1 10 QSIFSY LC CDR2 11 DAS LC CDR3 12 QQGYTRSDVDNA

TABLE 3 Amino acid sequences of variable regions for an anti-mesothelin antibody IgG SEQ mAb chain ID Amino acid sequence345A12- Heavy  13 QVQLVESGGGVVQPGRSLRLSCAASGIDL HC15-LC4 chainSSYAMSWVRQAPGKGLEWIGVIDISGNRF YADWVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARVDSRAWGPFNLWGQGTLVT VSS Light  14DYQMTQSPSSLSASVGDRVTITCQASQSI chain FSYLAWYQQKPGKAPKLLIYDASDLASGVPSRFSGSGSGTDFTLTISSLQCEDAATYY CQQGYTRSDVDNAFGGGTKVEIK

TABLE 4 Amino acid sequences of constant regions for an anti-mesothelin antibody Con- IgG stant SEQ mAb chain region IDAmino acid sequence 345A12- Heavy Human 15 ASTKGPSVFPLAPSSKSTSGGT HC15-chain IgG1 AALGCLVKDYFPEPVTVSWNSG LC4 ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGKLight Human 16 RTVAAPSVFIFPPSDEQLKSGT chain Ig ASVVCLLNNFYPREAKVQWKVD kappa NALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

TABLE 5 Amino acid sequences of full-length antibody Ig chains for an anti-mesothelin antibody IgG SEQ mAb chain IDAmino acid sequence 345A12- Heavy  17 QVQLVESGGGVVQPGRSLRLSCAASGIDLHC15- chain SSYAMSWVRQAPGKGLEWIGVIDISGNRF LC4YADWVKGRFTISRDNSKNTLYLQMSSLRA EDTAVYYCARVDSRAWGPFNLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK Light  18DYQMTQSPSSLSASVGDRVTITCQASQSI chain FSYLAWYQQKPGKAPKLLIYDASDLASGVPSRFSGSGSGTDFTLTISSLQCEDAATYY CQQGYTRSDVDNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

TABLE 6 Nucleic acid sequences encoding Kabat CDRs for an anti-mesothelin antibody IgG SEQ mAb chain IDNucleic acid sequence 345A12- HC CDR1 19 TCCTACGCCATGTCC HC15-LC4HC CDR2 20 GTGATCGACATCTCCGGCAACCGG TTCTACGCCGACTGGGTGAAGGGC HC CDR3 21GTGGACTCTAGAGCCTGGGGCCCC TTCAACCTG LC CDR1 22 CAGGCCTCCCAGTCCATCTTCTCCTACCTGGCC LC CDR2 23 GACGCCTCTGATCTGGCCTCC LC CDR3 24CAGCAGGGCTACACCAGATCCGAC GTGGACAACGCC

TABLE 7 Nucleic acid sequences encoding IMGT CDRs for an anti-mesothelin antibody IgG SEQ mAb chain IDNucleic acid sequence 345A12- HC CDR1 25 GGAATCGACCTGTCCTCCTACGCCHC15-LC4 HC CDR2 26 ATCGACATCTCCGGCAACCGG HC CDR3 27GCCAGAGTGGACTCTAGAGCCTGG GGCCCCTTCAACCTG LC CDR1 28 CAGTCCATCTTCTCCTACLC CDR2 29 GACGCCTCT LC CDR3 30 CAGCAGGGCTACACCAGATCCGAC GTGGACAACGCC

TABLE 8 Nucleic acid sequences encoding variable regions for an anti-mesothelin antibody IgG SEQ mAb chain IDNucleic acid sequence 345A12- Heavy  31 CAGGTGCAGCTGGTGGAATCTGGTGGCGGAHC15- chain GTGGTGCAGCCTGGCAGATCCCTGAGACTG LC4TCTTGTGCCGCCTCCGGAATCGACCTGTCC TCCTACGCCATGTCCTGGGTGCGACAGGCTCCTGGCAAGGGCCTGGAATGGATCGGCGTG ATCGACATCTCCGGCAACCGGTTCTACGCCGACTGGGTGAAGGGCCGGTTCACCATCTCC AGAGACAACTCCAAGAACACCCTGTACCTCCAGATGTCCTCCCTGCGGGCCGAGGATACC GCCGTGTACTACTGCGCCAGAGTGGACTCTAGAGCCTGGGGCCCCTTCAACCTGTGGGGC CAGGGAACACTCGTGACCGTGTCCTCT Light  32GATTACCAGATGACCCAGTCCCCCTCCAGC chain CTGTCCGCTTCTGTGGGCGACAGAGTGACCATCACCTGTCAGGCCTCCCAGTCCATCTTC TCCTACCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGAC GCCTCTGATCTGGCCTCCGGCGTGCCCTCTAGATTCTCCGGCTCTGGCTCTGGCACCGAC TTTACCCTGACCATCAGCTCCCTCCAGTGCGAGGATGCCGCCACCTACTACTGCCAGCAG GGCTACACCAGATCCGACGTGGACAACGCCTTTGGCGGAGGCACCAAGGTGGAAATCAAA

TABLE 9 Nucleic acid sequences encoding constant regions for an anti-mesothelin antibody Con- IgG stant SEQ mAb chainregion ID Nucleic acid sequence 345A12- Heavy Human 33GCATCCACCAAGGGCCCATCGGTC HC15-LC4 chain IgG1 TTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAG CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGAC AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAG AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG CTGGACTCCGACGGCTCCTTCTTCTTATATTCAAAGCTCACCGTGGAC AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCC GGGAAATGA Light Human 34CGAACTGTGGCTGCACCATCTGTC chain Ig TTCATCTTCCCGCCATCTGATGAG kappaCAGTTGAAATCTGGAACTGCCTCT GTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG TGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGG GGAGAGTGTTGA

TABLE 10 Nucleic acid sequences encoding full-lengthantibody Ig chains for an anti-mesothelin antibody⁺ IgG SEQ mAb chain IDNucleic acid sequence 345A12- Heavy  35 CAGGTGCAGCTGGTGGAATCTGGTGGCHC15- chain GGAGTGGTGCAGCCTGGCAGATCCCTG LC4 AGACTGTCTTGTGCCGCCTCCGGAATCGACCTGTCCTCCTACGCCATGTCCTGG GTGCGACAGGCTCCTGGCAAGGGCCTGGAATGGATCGGCGTGATCGACATCTCC GGCAACCGGTTCTACGCCGACTGGGTGAAGGGCCGGTTCACCATCTCCAGAGAC AACTCCAAGAACACCCTGTACCTCCAGATGTCCTCCCTGCGGGCCGAGGATACC GCCGTGTACTACTGCGCCAGAGTGGACTCTAGAGCCTGGGGCCCCTTCAACCTG TGGGGCCAGGGAACACTCGTGACCGTGTCCTCTGCATCCACCAAGGGCCCATCG GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTG GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAA GTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTG ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCTTATATTCAAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAATGA Light  36 GATTACCAGATGACCCAGTCCCCCTCC chainAGCCTGTCCGCTTCTGTGGGCGACAGA GTGACCATCACCTGTCAGGCCTCCCAGTCCATCTTCTCCTACCTGGCCTGGTAT CAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCTCTGATCTG GCCTCCGGCGTGCCCTCTAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACC CTGACCATCAGCTCCCTCCAGTGCGAGGATGCCGCCACCTACTACTGCCAGCAG GGCTACACCAGATCCGACGTGGACAACGCCTTTGGCGGAGGCACCAAGGTGGAA ATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCC AGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGTTGA +Nucleic acid sequences listed do notinclude leader sequences.

In some embodiments, an antibody or antigen-binding fragment disclosedherein binds to human mesothelin and comprises three heavy chaincomplementarity determining regions (HCDRs) comprising amino acidsequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions(LCDRs) comprising amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ IDNO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabatnumbering system (Kabat, Sequences of Proteins of ImmunologicalInterest.

In some embodiments, an antibody or antigen-binding fragment disclosedherein binds to human mesothelin and comprises three heavy chaincomplementarity determining regions (HCDRs) comprising amino acidsequences of SEQ ID NO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions(LCDRs) comprising amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ IDNO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGTnumbering system.

In various embodiments, the anti-mesothelin antibody or antigen-bindingfragment comprises three heavy chain CDRs and three light chain CDRs,wherein the CDRs include no more than one, two, three, four, five, orsix amino acid additions, deletions or substitutions of HCDR1 (SEQ IDNO: 1 according to Kabat, or SEQ ID NO: 7 according to IMGT), HCDR2 (SEQID NO: 2 according to Kabat, or SEQ ID NO: 8 according to IMGT), HCDR3(SEQ ID NO: 3 according to Kabat, or SEQ ID NO: 9 according to IMGT);and LCDR1 (SEQ ID NO: 4 according to Kabat, or SEQ ID NO: 10 accordingto IMGT), LCDR2 (SEQ ID NO: 5 according to Kabat, or SEQ ID NO: 11according to IMGT), and LCDR3 (SEQ ID NO: 6 according to Kabat, or SEQID NO: 12 according to IMGT).

In some embodiments, the anti-mesothelin antibody or antigen-bindingfragment is humanized. In some embodiments, the anti-mesothelin antibodyor antigen-binding fragment contains minimal sequence derived from anon-human immunoglobulin and retains the reactivity of a non-human(e.g., rabbit) antibody while being less immunogenic in human. In someembodiments, the anti-mesothelin antibody or antigen-binding fragmentprovides one or more of improved stability, formulatability, bindingaffinity, therapeutic efficacy, and/or decreased aggregation levels ascompared to one or more alternate anti-mesothelin antibodies.

In various embodiments, the anti-mesothelin antibody or antigen-bindingfragment comprises a heavy chain variable region comprising an aminoacid sequence of SEQ ID NO: 13 or a sequence that is at least 90%identical to SEQ ID NO: 13, and/or a light chain variable regioncomprising an amino acid sequence of SEQ ID NO: 14 or a sequence that isat least 90% identical to SEQ ID NO: 14. In various embodiments, theanti-mesothelin antibody or antigen-binding fragment comprises a heavychain constant region comprising an amino acid sequence of SEQ ID NO: 15or a sequence that is at least 90% identical to SEQ ID NO: 15, and/or alight chain constant region comprising an amino acid sequence of SEQ IDNO: 16 or a sequence that is at least 90% identical to SEQ ID NO: 16.

In various embodiments, the anti-mesothelin antibody or antigen-bindingfragment comprises a heavy chain amino acid sequence of SEQ ID NO: 17 ora sequence that is at least 90% identical to SEQ ID NO: 17, and/or alight chain amino acid sequence of SEQ ID NO: 18 or a sequence that isat least 90% identical to SEQ ID NO: 18.

In some embodiments, the anti-mesothelin antibody or antigen-bindingfragment comprises a human IgG1 heavy chain constant domain and a humanIg kappa light chain constant domain.

In various embodiments, the anti-mesothelin antibody or antigen-bindingfragment comprises three heavy chain complementarity determining regions(HCDRs) comprising amino acid sequences encoded by nucleic acidsequences of SEQ ID NO: 19 (HCDR1), SEQ ID NO: 20 (HCDR2), and SEQ IDNO: 21 (HCDR3); and three light chain complementarity determiningregions (LCDRs) comprising amino acid sequences encoded by nucleic acidsequences of SEQ ID NO: 22 (LCDR1), SEQ ID NO: 23 (LCDR2), and SEQ IDNO: 24 (LCDR3), as defined by the Kabat numbering system; or three heavychain complementarity determining regions (HCDRs) comprising amino acidsequences encoded by nucleic acid sequences of SEQ ID NO: 25 (HCDR1),SEQ ID NO: 26 (HCDR2), and SEQ ID NO: 27 (HCDR3); and three light chaincomplementarity determining regions (LCDRs) comprising amino acidsequences encoded by nucleic acid sequences of SEQ ID NO: 28 (LCDR1),SEQ ID NO: 29 (LCDR2), and SEQ ID NO: 30 (LCDR3), as defined by the IMGTnumbering system.

In various embodiments, the anti-mesothelin antibody or antigen-bindingfragment comprises a heavy chain variable region comprising an aminoacid sequence encoded by the nucleic acid sequence of SEQ ID NO: 31, anda light chain variable region comprising an amino acid sequence encodedby the nucleic acid sequence of SEQ ID NO: 32.

In various embodiments, the anti-mesothelin antibody or antigen-bindingfragment comprises a heavy chain constant region comprising an aminoacid sequence encoded by the nucleic acid sequence of SEQ ID NO: 33 anda light chain constant region comprising an amino acid sequence encodedby the nucleic acid sequence of SEQ ID NO: 34.

In various embodiments, the anti-mesothelin antibody or antigen-bindingfragment comprises a heavy chain comprising an amino acid sequenceencoded by the nucleic acid sequence of SEQ ID NO: 35, and a light chaincomprising an amino acid sequence encoded by the nucleic acid sequenceof SEQ ID NO: 36.

In various embodiments, the anti-mesothelin antibody or antigen-bindingfragment is 345A12-HC15-LC4.

The anti-mesothelin antigen-binding domains described herein may beuseful alone (e.g., as an antibody or antigen-binding fragment), linkedto one or more additional agents (e.g., as ADCs), or as part of a largermacromolecule (e.g., a bispecific antibody or multispecific antibody).

In some embodiments, the antibody or antigen-binding fragment isconjugated to a therapeutic agent. In some embodiments, thechemotherapeutic agent is eribulin. In some embodiments, thechemotherapeutic agent is an eribulin dimer.

In some embodiments, the antibody or antigen-binding fragment is anantigen-binding domain in and/or is part of a bispecific ormultispecific antibody. In some embodiments, the bispecific ormultispecific antibody comprises an antigen-binding domain that iscapable of binding to mesothelin and comprises three HCDRs comprisingamino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), andSEQ ID NO: 3 (HCDR3); and three light chain complementarity determiningregions (LCDRs) comprising amino acid sequences of SEQ ID NO: 4 (LCDR1),SEQ ID NO:5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabatnumbering system; or three heavy chain complementarity determiningregions (HCDRs) comprising amino acid sequences of SEQ ID NO: 7 (HCDR1),SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3); and three light chaincomplementarity determining regions (LCDRs) comprising amino acidsequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ IDNO: 12 (LCDR3), as defined by the IMGT numbering system. In someembodiments, the multispecific antibody comprises one or more additionalantigen binding domains, e.g., for the same antigen (i.e., mesothelin)or for other antigens.

In some embodiments, an antigen-binding domain is an antigen-bindingfragment. In some embodiments, the antigen-binding domain and/orantigen-binding fragment is a single chain variable fragment (scFv) or aFab fragment.

In some embodiments, the antigen-binding domains (e.g., theanti-mesothelin antigen-binding domains) disclosed herein, for use aloneor as part of a larger macromolecule, may include further modifications(e.g., one or more amino acid substitutions, deletions, and/orinsertions) while retaining mesothelin-binding function.

Antibody-Drug Conjugates

Further provided herein, in various embodiments, are antibody-drugconjugate (ADC) compounds comprising a linker that attaches achemotherapeutic drug moiety, e.g., eribulin, to an anti-mesothelinantibody disclosed herein. Antibody-drug conjugate (ADC) compounds maybe represented by Formula I:Ab−(L−D)_(p)  (I)wherein Ab is an internalizing anti-mesothelin antibody disclosed hereinor an internalizing antigen-binding fragment thereof;D is eribulin;L is a cleavable linker that covalently attaches Ab to D; andp is an integer from 1 to 8.

The ADC compounds of the present disclosure include an antibody moiety(including an antigen-binding fragment thereof) conjugated (e.g.,covalently attached by a linker) to a drug moiety (e.g., an eribulin),wherein the drug moiety when not conjugated to an antibody moiety has acytotoxic or cytostatic effect. In various embodiments, the drug moietyexhibits reduced or no cytotoxicity when bound in a conjugate butresumes cytotoxicity after cleavage from the linker and antibody moiety.In various embodiments, the drug moiety exhibits reduced or no bystanderkilling when bound in a conjugate but exhibits increased bystanderkilling after cleavage from a conjugate.

In some embodiments, the ADC compounds disclosed herein can selectivelydeliver an effective dose of a drug moiety (e.g., eribulin) to cancercells or to tumor tissues that express an antigen targeted by theantibody moiety of the ADC (e.g., mesothelin). In some embodiments, thedisclosed ADC compounds specifically target a cancer by deliveringeribulin to cells or tissues that express mesothelin while sparingnormal cells or tissues that either do not express mesothelin or expressmesothelin at much lower levels. In some embodiments, the disclosed ADCcompounds have improved on-target killing and/or reduced off-targetkilling, as compared to an ADC comprising an alternate antibody, linker,and/or drug moiety, e.g., BAY 94-9343. In some embodiments, thedisclosed ADC compounds have improved ADCC activity retention by theADC, as compared to an ADC comprising an alternate antibody, linker,and/or drug moiety, e.g., BAY 94-9343. In some embodiments, thedisclosed ADC compounds have improved stability (e.g., plasmastability), as compared to an ADC comprising an alternate antibody,linker, and/or drug moiety, e.g., BAY 94-9343. In some embodiments, thedisclosed ADC compounds have improved anti-tumor efficacy, as comparedto an ADC comprising an alternate antibody, linker, and/or drug moiety,e.g., BAY 94-9343.

In some embodiments, the ADC compounds disclosed herein can providefavorable anti-tumor efficacy with a lower dose of eribulin, as comparedto the dose of eribulin when evaluated as a stand-alone drug (i.e., notconjugated to an antibody moiety). In some embodiments, thetumor-specific targeting of the ADC compounds disclosed herein increasesanti-tumor activity and/or decreases off-target cytotoxicity of the ADC,as compared to eribulin when evaluated as a stand-alone drug. Forinstance, in some embodiments, the ADC compounds disclosed herein showfavorable anti-tumor activity with a dose of eribulin that is at least10-fold lower, at least 15-fold lower, at least 20-fold lower, or atleast 30-fold lower than the dose of eribulin when evaluated as astand-alone drug. In some embodiments, the disclosed ADC compoundsdemonstrate anti-tumor activity that is comparable to or superior to theactivity of eribulin when evaluated as a stand-alone drug, whileproviding an improved toxicologic or safety profile over that of theeribulin on its own.

In some embodiments, the linker is stable outside a cell, such that theADC remains intact when present in extracellular conditions but iscapable of being cleaved on internalization in a cell, e.g., a cancercell. In some embodiments, an eribulin drug moiety is cleaved from ananti-mesothelin antibody moiety when the ADC enters a cell thatexpresses mesothelin, and cleavage releases an unmodified form oferibulin.

In some embodiments, the linker comprises a cleavable moiety that ispositioned such that no part of the linker or the antibody moietyremains bound to the eribulin drug moiety upon cleavage. In someembodiments, the cleavable moiety in the linker is a cleavable peptidemoiety. In some embodiments, an ADC that comprises a cleavable peptidemoiety demonstrates lower aggregation levels, improved antibody:drugratio, increased on-target killing of cancer cells, decreased off-targetkilling of non-cancer cells, and/or higher drug loading (p) relative toan ADC that comprises an alternate linker moiety. In some embodiments,the increased potency and/or cytotoxicity is provided in a cancerexpressing moderate levels of mesothelin. In some embodiments, thecleavable peptide moiety is cleavable by an enzyme, and the linker is anenzyme-cleavable linker. In some embodiments, the enzyme is cathepsin B,and the linker is a cathepsin-cleavable linker. In some embodiments, theenzyme-cleavable linker (e.g., the cathepsin-cleavable linker) exhibitsone or more of the improved properties mentioned above, as compared toan alternate cleavage mechanism.

In some embodiments, the cleavable peptide moiety in the linkercomprises an amino acid unit. In some embodiments, the amino acid unitcomprises valine-citrulline (Val-Cit). In some embodiments, an ADC thatcomprises Val-Cit demonstrates increased stability, decreased off-targetcell killing, increased on-target cell killing, lower aggregationlevels, and/or higher drug loading relative to an ADC that comprises analternate amino acid unit or alternate cleavable moiety.

In some embodiments, the linker comprises at least one spacer unitjoining the antibody moiety to the cleavable moiety. In someembodiments, the spacer unit in the linker may comprise at least onepolyethylene glycol (PEG) moiety. The PEG moiety may, for example,comprise —(PEG)_(m)-, wherein m is an integer from 1 to 10. In someembodiments, the spacer unit in the linker comprises (PEG)₂. In someembodiments, an ADC that comprises a shorter spacer unit (e.g., (PEG)₂)demonstrates lower aggregation levels and/or higher drug loadingrelative to an ADC that comprises a longer spacer unit (e.g., (PEG)₈)despite the shorter linker length.

In some embodiments, the spacer unit in the linker attaches to theantibody moiety of the ADC via a maleimide moiety (Mal). In someembodiments, an ADC that comprises a linker attached to the antibodymoiety via a Mal demonstrates higher drug loading relative to an ADCthat comprises a linker attached to the antibody moiety via an alternatemoiety. In some embodiments, the Mal in the linker is joined to theantibody moiety via a cysteine residue (e.g., LCcys80). In someembodiments, the Mal in the linker is joined to a cysteine residue(e.g., LCcys80) of a light chain variable region on the antibody orantigen-binding fragment. In some embodiments, p is 2 and two -L-Dmoieties are attached to the antibody or antigen-binding fragment. Insome embodiments, each -L-D moiety is attached to a cysteine residue(e.g., LCcys80) of a light chain variable region on the antibody orantigen-binding fragment. In some embodiments, the cysteine residue is aLCcys80. In some embodiments, the Mal-spacer unit comprises a PEGmoiety. In some embodiments, the linker comprises Mal-(PEG)_(m), e.g.,Mal-(PEG)₂. In some embodiments, the Mal-spacer unit attaches theantibody moiety to the cleavable moiety in the linker. In someembodiments, the cleavable moiety in the linker is a cleavable peptidemoiety, e.g., an amino acid unit. In some embodiments, the linkercomprises Mal-(PEG)₂-Val-Cit.

In some embodiments, the cleavable moiety in the linker is directlyjoined to the eribulin drug moiety of the ADC, and the cleavable moietyis either directly connected to the antibody moiety or connected througha spacer unit. In some embodiments, a spacer unit also attaches thecleavable moiety in the linker to the eribulin drug moiety. In someembodiments, the spacer unit that attaches the cleavable moiety in thelinker to the eribulin drug moiety is self-immolative. In someembodiments, the self-immolative spacer is capable of releasingunmodified eribulin in a target cell. In some embodiments, theself-immolative spacer unit comprises a p-aminobenzyl alcohol e.g.,p-aminobenzyloxycarbonyl (pAB). The pAB in the linker, in someembodiments, attaches the cleavable moiety to the eribulin drug moiety.In some embodiments, the cleavable moiety is a cleavable peptide moiety,e.g., an amino acid unit. In some embodiments, the linker comprisesVal-Cit-pAB. In some embodiments, the linker comprises Val-Cit-pAB and aPEG spacer unit joining the linker to the antibody moiety through a Mal.

In some embodiments, p is an integer from 1 to 8, or from 2 to 6. Insome embodiments, p is 2 or 6. In some embodiments, the linker comprisesMal-(PEG)₂-Val-Cit-pAB. In some embodiments, the linker comprisesMal-(PEG)₂-Val-Cit-pAB and p is 2. In some embodiments, the linkercomprises Mal-(PEG)₂-Val-Cit-pAB and p is 6.

In some embodiments, the antibody moiety is conjugated to eribulin drugmoiety via a linker comprising a Mal moiety, a PEG moiety, Val-Cit, anda pAB. In these embodiments, the maleimide moiety covalently attachesthe linker-drug moiety to the antibody moiety, and the pAB acts as aself-immolative spacer unit. Such linker may be referred to as the“Mal-VC-pAB” linker, the “Mal-VCP”, “maleimide-VCP”, or “VCP” linker,the “Mal-(PEG)₂-VCP” linker, or the “Mal-(PEG)₂-Val-Cit-pAB” linker. Insome embodiments, the eribulin drug moiety is eribulin covalently linkedat the C-35 position. In some embodiments, the pAB of theMal-(PEG)₂-Val-Cit-pAB linker is attached to the C-35 amine on theeribulin drug moiety.

345A12-HC15-LC4 is an exemplary anti-mesothelin antibody comprising orencoded by the sequences shown above in Tables 1-10, e.g., comprising aheavy chain variable region comprising an amino acid sequence of SEQ IDNO: 13 and a light chain variable region comprising an amino acidsequence of SEQ ID NO: 14. In some embodiments, the antibody moiety ofthe ADCs disclosed herein comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO: 13 and a light chainvariable region comprising an amino acid sequence of SEQ ID NO: 14. Insome embodiments, the antibody moiety of the ADCs disclosed herein is345A12-HC15-LC4.

In some embodiments, an ADC disclosed herein comprises345A12-HC15-LC4-VCP-eribulin. In these embodiments, an antibody moietycomprising 345A12-HC15-LC4 is joined to an eribulin drug moiety via alinker comprising Mal-(PEG)₂-Val-Cit-pAB. Such ADC may be referred to as“MORAb-109.” In some embodiments, an ADC disclosed herein is MORAb-109.

In some embodiments, an ADC disclosed herein is MORAb-109 and has a p of2. In some embodiments, when p is 2, the ADC may be referred to as“MORAb-109 (DAR2).” In other embodiments, an ADC disclosed herein isMORAb-109 and has a p of 6. In some embodiments, when p is 6, the ADCmay be referred to as “MORAb-109 (DAR6).”

In various embodiments, the linker is designed to facilitate bystanderkilling (the killing of neighboring cells, e.g., those that do notexpress mesothelin) through cleavage after cellular internalization anddiffusion of the linker-drug moiety and/or the drug moiety alone toneighboring cells. In some embodiments, the linker promotes cellularinternalization. In some embodiments, the linker is designed to minimizecleavage in the extracellular environment and thereby reduce toxicity tooff-target tissue (e.g., non-cancerous tissue), while preserving ADCbinding to target tissue and bystander killing of cancerous tissue thatdoes not express an antigen targeted by the antibody moiety of an ADC,but surrounds target cancer tissue expressing that antigen. In someembodiments, a linker comprising a maleimide (Mal) moiety, apolyethylene glycol (PEG) moiety, valine-citrulline (Val-Cit or “VC”),and a pAB provides these functional features. In some embodiments, alinker comprising Mal-(PEG)₂-Val-Cit-pAB is particularly effective inproviding these functional features when joining an antibody moiety andan eribulin drug moiety. In some embodiments, a linker comprisingMal-(PEG)₂-Val-Cit-pAB is effective in providing some or all of thesefunctional features when joining an anti-mesothelin antibody moiety suchas 345A12-HC15-LC4 and an eribulin drug moiety.

In some embodiments, an anti-mesothelin antibody or antigen-bindingfragment comprises sequences disclosed herein (e.g., comprising the sixCDRs and/or heavy and light chain variable domains disclosed in Tables1-3). In some embodiments, the antibody or antigen-binding fragment is afull-length antibody. In some embodiments, the antibody orantigen-binding fragment is a monospecific antibody or antigen-bindingfragment, a bispecific antibody or antigen-binding fragment, or amultispecific antibody or antigen-binding fragment. In some embodiments,the antibody or antigen-binding fragment is a single chain variablefragment (scFv) or a Fab fragment.

In some embodiments, an ADC comprising an anti-mesothelin antibody (Ab)moiety and a cleavable peptide moiety demonstrates lower aggregationlevels, improved antibody:drug ratio, increased on-target killing ofcancer cells, decreased off-target killing of non-cancer cells, higherdrug loading (p), increased cytotoxicity, and/or potency relative to anADC comprising an alternate antibody or antigen-binding fragment. Insome embodiments, the ADC is an ADC of Formula (I):Ab−(L−D)_(p)  (I)wherein Ab is an antibody or antigen-binding fragment, wherein theantibody or antigen-binding fragment is capable of binding to mesothelinand comprises three heavy chain complementarity determining regions(HCDRs) comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ IDNO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3); and three light chaincomplementarity determining regions (LCDRs) comprising amino acidsequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO:6 (LCDR3), as defined by the Kabat numbering system; or three heavychain complementarity determining regions (HCDRs) comprising amino acidsequences of SEQ ID NO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions(LCDRs) comprising amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ IDNO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGTnumbering system;D is a chemotherapeutic agent (e.g., eribulin);L is a cleavable linker that covalently attaches Ab to D; andp is an integer from 1 to 8.

In some embodiments, the antibody or antigen-binding fragment comprisesa heavy chain variable region comprising an amino acid sequence of SEQID NO: 13, and a light chain variable region comprising an amino acidsequence of SEQ ID NO: 14. In some embodiments, the antibody orantigen-binding fragment comprises a human IgG1 heavy chain constantdomain and a human Ig kappa light chain constant domain. In someembodiments, the antibody or antigen-binding fragment comprises a heavychain constant region comprising an amino acid sequence of SEQ ID NO:15, and a light chain constant region comprising an amino acid sequenceof SEQ ID NO: 16. In some embodiments, the antibody or antigen-bindingfragment comprises a heavy chain comprising an amino acid sequence ofSEQ ID NO: 17, and a light chain comprising an amino acid sequence ofSEQ ID NO: 18.

In some embodiments, the ADC has Formula (I):Ab−(L−D)_(p)  (I)wherein:Ab is an antibody or antigen-binding fragment, wherein the antibody orantigen-binding fragment is capable of binding to mesothelin and/or amesothelin-expressing cell and comprises three heavy chaincomplementarity determining regions (HCDRs) comprising amino acidsequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions(LCDRs) comprising amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ IDNO:5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabatnumbering system; or three heavy chain complementarity determiningregions (HCDRs) comprising amino acid sequences of SEQ ID NO: 7 (HCDR1),SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3); and three light chaincomplementarity determining regions (LCDRs) comprising amino acidsequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ IDNO: 12 (LCDR3), as defined by the IMGT numbering system;D is an eribulin;L is a cleavable linker that covalently attaches Ab to D; andp is an integer from 1 to 8.

In some embodiments, the antibody or antigen-binding fragment thattargets mesothelin and/or a mesothelin-expressing cell comprises a heavychain variable region comprising an amino acid sequence of SEQ ID NO:13, and a light chain variable region comprising an amino acid sequenceof SEQ ID NO: 14. In some embodiments, the antibody or antigen-bindingfragment comprises a human IgG1 heavy chain constant domain and a humanIg kappa light chain constant domain. In some embodiments, the antibodyor antigen-binding fragment comprises a heavy chain constant regioncomprising an amino acid sequence of SEQ ID NO: 15, and a light chainconstant region comprising an amino acid sequence of SEQ ID NO: 16. Insome embodiments, the antibody or antigen-binding fragment comprises aheavy chain comprising an amino acid sequence of SEQ ID NO: 17, and alight chain comprising an amino acid sequence of SEQ ID NO: 18.

In some embodiments, the ADC has Formula (I):Ab−(L−D)_(p)  (I)wherein:Ab is an antibody or antigen-binding fragment thereof that targetsmesothelin and/or a mesothelin-expressing cell comprising three heavychain complementarity determining regions (HCDRs) comprising amino acidsequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions(LCDRs) comprising amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ IDNO:5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabatnumbering system; or three heavy chain complementarity determiningregions (HCDRs) comprising amino acid sequences of SEQ ID NO: 7 (HCDR1),SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3); and three light chaincomplementarity determining regions (LCDRs) comprising amino acidsequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ IDNO: 12 (LCDR3), as defined by the IMGT numbering system;D is an eribulin;L is a cleavable linker comprising Mal-(PEG)₂-Val-Cit-pAB; andp is an integer from 1 to 8.

In some embodiments, the antibody or antigen-binding fragment thattargets mesothelin and/or a mesothelin-expressing cell comprises a heavychain variable region comprising an amino acid sequence of SEQ ID NO:13, and a light chain variable region comprising an amino acid sequenceof SEQ ID NO: 14. In some embodiments, the antibody or antigen-bindingfragment comprises a human IgG1 heavy chain constant domain and a humanIg kappa light chain constant domain. In some embodiments, the antibodyor antigen-binding fragment comprises an IgG1 heavy chain constantregion comprising an amino acid sequence of SEQ ID NO: 15, and an Igkappa light chain constant region comprising an amino acid sequence ofSEQ ID NO: 16. In some embodiments, the antibody or antigen-bindingfragment comprises a heavy chain comprising an amino acid sequence ofSEQ ID NO: 17, and a light chain comprising an amino acid sequence ofSEQ ID NO: 18.

In some embodiments, the ADC has Formula (I):Ab−(L−D)_(p)  (I)wherein:Ab is an antibody or antigen-binding fragment, wherein the antibody orantigen-binding fragment is capable of binding to mesothelin andcomprises a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 13, and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 14;D is an eribulin;L is a cleavable linker comprising Mal-(PEG)₂-Val-Cit-pAB; andp is an integer from 1 to 8.

In some embodiments, the antibody or antigen-binding fragment comprisesa human IgG1 heavy chain constant region, and a human Ig kappa lightchain constant region. In some embodiments, the antibody orantigen-binding fragment comprises a heavy chain constant regioncomprising an amino acid sequence of SEQ ID NO: 15, and a light chainconstant region comprising an amino acid sequence of SEQ ID NO: 16. Insome embodiments, the antibody or antigen-binding fragment comprises aheavy chain comprising an amino acid sequence of SEQ ID NO: 17, and alight chain comprising an amino acid sequence of SEQ ID NO: 18.

In some embodiments, the antibody or antigen-binding fragment of the ADCis 345A12-HC15-LC4. In some embodiments, p is 1 to 8. In someembodiments, p is 2 or 6. In some embodiments, p is 2.

In some embodiments, an ADC disclosed herein (e.g., one comprising ananti-mesothelin antibody and linker disclosed herein) with a lower levelof eribulin drug loading (e.g., a p of 2) can deliver the same orsimilar levels of eribulin to a cancer cell or to a tumor tissue as anADC with a higher level of drug loading (e.g., a p of 6). In someembodiments, an ADC with a lower level of drug loading (e.g., a p of 2)can provide tumor growth inhibition and/or in vivo anti-cancer treatmentefficacy approximately comparable to or superior to that of an ADC witha higher level of drug loading (e.g., a p of 6).

In some embodiments, each eribulin moiety is joined by a cleavablelinker to the mesothelin-targeting antibody or antigen-binding fragmentvia a cysteine residue on the antibody or fragment (e.g., LCcys80). Insome embodiments, a total of two linker-eribulin moieties are attachedto the mesothelin-targeting antibody or antigen-binding fragment, e.g.,via two cysteine residues on the antibody or antigen-binding fragment(i.e., such that the ADC has a DAR2). In some embodiments, the cysteineresidue(s) is/are LCcys80.

The development and production of an ADC for use as a human therapeuticagent, e.g., as an oncologic agent, may require more than theidentification of an antibody capable of binding to a desired target ortargets and attaching to a drug used on its own to treat cancer. Linkingthe antibody to the drug may have significant and unpredictable effectson the activity of one or both the antibody and the drug, effects whichwill vary depending on the antibody and/or type of linker and/or drugchosen. In some embodiments, therefore, the components of the ADC areselected to (i) retain one or more therapeutic properties exhibited bythe antibody and drug moieties in isolation, (ii) maintain the specificbinding properties of the antibody moiety; (iii) optimize drug loadingand drug-to-antibody ratios; (iv) allow delivery, e.g., intracellulardelivery, of the drug moiety via stable attachment to the antibodymoiety; (v) retain ADC stability as an intact conjugate until transportor delivery to a target site; (vi) minimize aggregation of the ADC priorto or after administration; (vii) allow for the therapeutic effect,e.g., cytotoxic effect, of the drug moiety after cleavage in thecellular environment; (viii) exhibit in vivo anti-cancer treatmentefficacy comparable to or superior to that of the antibody and drugmoieties in isolation; (ix) minimize off-target killing by the drugmoiety; and/or (x) exhibit desirable pharmacokinetic andpharmacodynamics properties, formulatability, andtoxicologic/immunologic profiles. Screening each of these properties maybe needed to identify an improved ADC for therapeutic use (Ab et al.(2015) Mol. Cancer Ther. 14:1605-13).

In some embodiments, an ADC disclosed herein comprising ananti-mesothelin antibody or antigen-binding fragment joined to achemotherapeutic, e.g., eribulin, demonstrates a particular combinationof desirable properties. These properties include, but are not limitedto, effective levels of drug loading, low aggregation levels, stabilityunder storage conditions and/or when in circulation in the body (e.g.,serum and matrix stability), retained affinity for target-expressingcells comparable to unconjugated antibody, potent cytotoxicity againsttarget-expressing cells, high levels of bystander killing, and/oreffective in vivo anti-cancer activity, all as compared to ADCs usingother antibody moieties. In some embodiments, the high anti-canceractivities of these conjugates are seen even when tested in cell lineshaving moderate antigen expression, demonstrating potent sensitivity totoxin payload delivered by the ADC. In some embodiments, an ADCcomprising an anti-mesothelin antibody or antigen-binding fragmentdisclosed herein exhibits particularly favorable anti-tumor cytotoxicityand/or potency, and improved off-target toxicity and drug metabolism andpharmacokinetic (DMPK) profiles as compared to an ADC comprising analternate antibody moiety. In some embodiments, an ADC comprising ahumanized anti-mesothelin antibody disclosed herein and eribulinprovides surprisingly favorable pharmacological and toxicologicalproperties as compared to an ADC comprising an alternate antibody moietyand/or conjugate.

The ADC compounds of the present disclosure may selectively deliver aneffective dose of a cytotoxic or cytostatic agent to cancer cells or totumor tissue. In some embodiments, the cytotoxic and/or cytostaticactivity of the ADC is dependent on the target antigen expression levelin a cell. In some embodiments, the disclosed ADCs are particularlyeffective at killing cancer cells expressing a high level of targetantigen, as compared to cancer cells expressing the same antigen at alow level. In some embodiments, the disclosed ADCs are particularlyeffective at killing cancer cells expressing the target antigen at amoderate level, as compared to cancer cells expressing the same antigenat a low level.

Exemplary high mesothelin-expressing cancers include but are not limitedto ovarian cancer (e.g., serous ovarian cancer, clear cell ovariancancer), pancreatic cancer, mesothelioma, endometrial cancer, non-smallcell lung cancer (e.g., adenocarcinoma), and colorectal cancer.Exemplary moderate mesothelin-expressing cancers include but are notlimited to gastric cancer, thymic carcinoma, and cholangiocellularcarcinoma. Exemplary low mesothelin-expressing cancers include but arenot limited to melanoma and lymphoma. In some embodiments,mesothelin-expressing cancers may include cancers harboring mutationsand/or drug resistance, e.g., KRAS/STK11 mutated lung cancer (non-smallcell lung adenocarcinoma), for example those mutated lung cancers thatexhibit resistance to treatment with PD-1 checkpoint blockade.

Drug Moieties

The drug moiety (D) of the ADCs described herein can be anychemotherapeutic agent. Useful classes of chemotherapeutic agentsinclude, for example, anti-tubulin agents. In certain embodiments, thedrug moiety is an anti-tubulin agent. One exemplary drug moiety for usein the described ADCs and compositions is eribulin. Another exemplarydrug moiety for use in the described ADCs and compositions is aneribulin dimer.

In various embodiments, the structure of eribulin used in its naturalform in the disclosed ADCs is as shown in Formula (II):

In various other embodiments, the structure of the eribulin used in thedisclosed ADCs is as shown in Publ. No. US 20180193478, which isincorporated herein by reference for all eribulin structures and methodsof synthesizing those structures.

Drug Loading

Drug loading may be represented by p, and is also referred to herein asthe drug-to-antibody ratio (DAR). Drug loading may range from, e.g., 1to 10 drug moieties per antibody moiety. In some embodiments, p is aninteger from 1 to 10. In some embodiments, p is an integer from 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Insome embodiments, p is an integer from 2 to 10, 2 to 9, 2 to 8, 2 to 7,2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is an integerfrom 1 to 8. In some embodiments, p is an integer from 1 to 6. In someembodiments, p is an integer from 2 to 6. In some embodiments, p is 2.In some embodiments, p is 6.

Drug loading may be limited, in some embodiments, by the number ofattachment sites on the antibody moiety. In some embodiments, the linkermoiety (L) of the ADC attaches to the antibody moiety through achemically active group on one or more amino acid residues on theantibody moiety. For example, the linker may be attached to the antibodymoiety via a free amino, imino, hydroxyl, thiol, or carboxyl group(e.g., to the N- or C-terminus, to the epsilon amino group of one ormore lysine residues, to the free carboxylic acid group of one or moreglutamic acid or aspartic acid residues, or to the sulfhydryl group ofone or more cysteine residues). The site to which the linker is attachedcan be a natural residue in the amino acid sequence of the antibodymoiety, or it can be introduced into the antibody moiety, e.g., by DNArecombinant technology (e.g., by introducing a cysteine residue into theamino acid sequence) or by protein biochemistry (e.g., by reduction, pHadjustment, or hydrolysis).

In some embodiments, the number of drug moieties that can be conjugatedto an antibody moiety is limited by the number of free cysteineresidues. For example, where the attachment is a cysteine thiol group,an antibody may have only one or a few cysteine thiol groups, or mayhave only one or a few sufficiently reactive thiol groups through whicha linker may be attached. Generally, antibodies do not contain many freeand reactive cysteine thiol groups that may be linked to a drug moiety.Indeed, most cysteine thiol residues in antibodies are involved ineither interchain or intrachain disulfide bonds. Conjugation tocysteines can therefore, in some embodiments, require at least partialreduction of the antibody. Over-attachment of linker-toxin to anantibody may destabilize the antibody by reducing the cysteine residuesavailable to form disulfide bonds. Thus, in some embodiments, an optimaldrug:antibody ratio should increase potency of the ADC (by increasingthe number of attached drug moieties per antibody) without destabilizingthe antibody moiety. In some embodiments, an optimal ratio may be 2 or6. In some embodiments, an optimal ratio is 2.

In some embodiments, one or more site-specific conjugation technologiesare used to produce a homogeneous ADC product with a defined drugloading, i.e., a defined drug-to-antibody ratio (DAR). In someembodiments, free cysteine residues can be generated in the light chainor heavy chain of antibodies for site-specific conjugation viaResidue-SPEcific Conjugation Technology (RESPECT). Exemplary protocolsfor the generation of RESPECT-formatted antibodies are described inAlbone et al. (2017) Cancer Biol. Ther. 18(5):347-57, and in Intl. Pub.Nos. WO/2016205618 and WO/2017106643, each of which is incorporatedherein by reference for methods of performing site-specific conjugation.In some embodiments, an ADC is produced using site-specific conjugationto covalently attach an antibody moiety to a drug moiety via a linker(e.g., a Mal-(PEG)₂-Val-Cit-pAB linker). In some embodiments,site-specific conjugation is used to target a DAR of about 2 for ADCs orcompositions comprising an eribulin drug moiety.

Rabbit monoclonal antibodies chimerized or humanized to human constantregions may produce unpaired cysteines within the light chain, leavingthose residues available for conjugation (Albone et al. (2017) CancerBiol. Ther. 18(5):347-57; Intl. Pub. No. WO/2016205618). In someembodiments, the antibody moiety used for site-specific conjugation is aRESPECT-L-formatted antibody. Exemplary RESPECT-L-formatted antibodieswith an unpaired cysteine at light chain position 80 (LCcys80) aredescribed herein. As used herein, “LCcys80” or “Cys80” refers to acysteine residue at amino acid position 80 of a light chain variableregion on an antibody or an antigen-binding fragment according to theKabat numbering system. For example, in some embodiments, in the lightchain variable regions disclosed herein, LCcys80 occurs at amino acidposition 80. RESPECT-L-derived antibodies can yield an ADC with a DAR ofabout 2. In some embodiments, a drug loading and/or an average drugloading of about 2 is achieved, e.g., using site-specific conjugation.

Pharmaceutical Compositions

In some embodiments, the present disclosure further providespharmaceutical compositions comprising one or more antibodies,antigen-binding fragments, conjugates, and/or ADCs disclosed herein anda pharmaceutically acceptable carrier. In some embodiments, thepharmaceutical compositions described herein comprise at least oneadditional agent.

In some embodiments, the present disclosure further providespharmaceutical compositions comprising multiple copies of an antibody,antigen-binding fragment, conjugate, and/or ADC disclosed herein. Insome embodiments, the present disclosure further provides pharmaceuticalcompositions comprising multiple copies of an ADC disclosed herein. Insome embodiments, the average p of the ADCs in a composition is fromabout 1 to about 8. In some embodiments, the average p of the ADCs inthe composition is about 2 or about 6. In some embodiments, the averagep of the ADCs in the composition is about 1.3, about 1.4, about 1.5,about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about2.2, or about 2.3. In some embodiments, the average p of the ADCs in thecomposition is about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,or 6.5.

In some embodiments, a pharmaceutical composition may further compriseone or more additional therapeutic agents, e.g., one or more agentscapable of treating a mesothelin-expressing cancer, a steroid, and thelike.

Therapeutic Uses and Methods of Treatment

Disclosed herein are methods of using the disclosed antibodies,antigen-binding fragments, conjugates, ADCs and/or pharmaceuticalcompositions in treating a subject for a disorder, e.g., an oncologicdisorder. The antibodies, antigen-binding fragments, conjugates, and/orADCs may be administered alone or in combination with a secondtherapeutic agent, and may be administered in any pharmaceuticallyacceptable formulation, dosage, and dosing regimen. The antibodies,antigen-binding fragments, and/or ADC treatment efficacy may beevaluated for toxicity as well as indicators of efficacy and adjustedaccordingly. Efficacy measures include, but are not limited to, acytostatic and/or cytotoxic effect observed in vitro or in vivo, reducedtumor volume, tumor growth inhibition, and/or prolonged survival.

Methods of determining whether an antibody, antigen-binding fragment,and/or ADC exerts a cytostatic and/or cytotoxic effect on a cell areknown. For example, the cytotoxic or cytostatic activity of an antibody,antigen-binding fragment, and/or ADC can be measured by exposingmammalian cells expressing a target protein of the antibody,antigen-binding fragment, and/or ADC in a cell culture medium; culturingthe cells for a period from about 6 hours to about 5 days; and measuringcell viability. Cell-based in vitro assays may also be used to measureviability (proliferation), cytotoxicity, and induction of apoptosis(caspase activation) of the ADC.

For determining whether an antibody, antigen-binding fragment, and/orADC exerts a cytostatic effect, a thymidine incorporation assay may beused. For example, cancer cells expressing a target antigen at a densityof 5,000 cells/well of a 96-well plated can be cultured for a 72-hourperiod and exposed to 0.5 μCi of 3H-thymidine during the final 8 hoursof the 72-hour period. The incorporation of 3H-thymidine into cells ofthe culture is measured in the presence and absence of the antibody,antigen-binding fragment, and/or ADC.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) may be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that an ADC is useful in the treatment of cancers.

Cell viability may be measured, e.g., by determining in a cell theuptake of a dye such as neutral red, trypan blue, Crystal Violet, orALAMAR™ blue (see, e.g., Page et al. (1993) Intl. J. Oncology 3:473-6).In such an assay, the cells are incubated in media containing the dye,the cells are washed, and the remaining dye, reflecting cellular uptakeof the dye, is measured spectrophotometrically. In certain embodiments,in vitro potency and/or cytotoxicity of prepared ADCs is assessed usinga Crystal Violet assay. Crystal Violet is a triarylmethane dye thataccumulates in the nucleus of viable cells. In this assay, cells areexposed to the ADCs or control agents for a defined period of time,after which, cells are stained with Crystal Violet, washed copiouslywith water, then solubilized with 1% SDS and readspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytotoxicity (Skehan et al. (1990) J. Natl.Cancer Inst. 82:1107-12).

Apoptosis can be quantitated, for example, by measuring DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica (1999) No. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis may also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal., eds. (1992) pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The disclosed ADCs may also be evaluated for bystander killing activity.Bystander killing activity may be determined, e.g., by an assayemploying two cell lines, one positive for target antigen and onenegative for target antigen. The cell lines may be labeled todifferentiate them. For example, target-positive cells labeled withNuclight™ Green (NLG) and target-negative cells labeled with Nuclight™Red (NLR) may be co-cultured, treated with an ADC followed by monitoringof cytotoxicity. Killing of the target-negative cells when mixed withtarget-positive cells is indicative of bystander killing, whereaskilling of the target-negative cells in the absence of thetarget-positive cells is indicative of off-target killing.

In some embodiments, the present disclosure features a method ofkilling, inhibiting or modulating the growth of, or interfering with themetabolism of, a cancer cell or tissue by disrupting tubulin. The methodmay be used with any subject where disruption of tubulin provides atherapeutic benefit. Subjects that may benefit from disrupting tubulininclude, but are not limited to, those having or are at risk of having agastric cancer, ovarian cancer (e.g., epithelial ovarian cancer), lungcancer (e.g., non-small cell lung cancer), breast cancer, endometrialcancer (e.g., serous endometrial carcinoma), osteosarcoma, Kaposi'ssarcoma, testicular germ cell cancer, head and neck cancer, livercancer, renal cancer, urothelial cancer, uterine cancer, bile ductcancer, leukemia (e.g., acute myeloid leukemia), lymphoma (e.g.,Hodgkin's disease, non-Hodgkin's lymphoma), myeloma, head and neckcancer, esophageal cancer, pancreatic cancer, prostate cancer, braincancer (e.g., glioblastoma), thyroid cancer, colorectal cancer, and/orskin cancer (e.g., melanoma), or any metastases thereof (Dumontet andJordan (2010) Nat. Rev. Drug Discov. 9:790-803).

In various embodiments, the disclosed antibodies, antigen-bindingfragments, and/or ADCs may be administered in any cell or tissue thatexpresses mesothelin, such as a mesothelin-expressing cancer cell ortissue. An exemplary embodiment includes a method of inhibitingmesothelin-mediated cell signaling or a method of killing a cell. Themethod may be used with any cell or tissue that expresses mesothelin,such as a cancerous cell or a metastatic lesion. Non-limiting examplesof mesothelin-expressing cancers include mesothelioma, pancreatic cancer(e.g., pancreatic adenocarcinoma), ovarian cancer (e.g., serous ovariancancer, clear cell ovarian cancer, epithelial ovarian cancer), and lungcancer (e.g., non-small cell lung cancer, lung adenocarcinoma) (Wang etal. (2012) PLoS ONE 7:e33214). Other exemplary mesothelin-cancersinclude endometrial cancer, colorectal cancer, gastric cancer, leukemia,breast cancer, cervical cancer, head and neck cancer, liver cancer,prostate cancer, renal cancer, thyroid cancer, urothelial cancer,uterine cancer, and bile duct cancer. Non-limiting examples ofmesothelin-expressing cells include OVCAR3 human ovarian carcinomacells, HEC-251 human endometrioid cells, H226 human lung squamous cellmesothelioma cells, and cells comprising a recombinant nucleic acidencoding mesothelin or a portion thereof.

Exemplary methods include the steps of contacting a cell with anantibody, antigen-binding fragment, and/or ADC, as described herein, inan effective amount, i.e., amount sufficient to kill the cell. Themethod can be used on cells in culture, e.g., in vitro, in vivo, exvivo, or in situ. For example, cells that express mesothelin (e.g.,cells collected by biopsy of a tumor or metastatic lesion; cells from anestablished cancer cell line; or recombinant cells), can be cultured invitro in culture medium and the contacting step can be affected byadding the antibody, antigen-binding fragment, and/or ADC to the culturemedium. The method will result in killing of cells expressingmesothelin, including in particular tumor cells expressing mesothelin.Alternatively, the antibody, antigen-binding fragment, and/or ADC can beadministered to a subject by any suitable administration route (e.g.,intravenous, subcutaneous, or direct contact with a tumor tissue) tohave an effect in vivo. This approach can also be used for antibodiesand ADCs targeting other cell surface antigens.

The in vivo effect of a disclosed antibody, antigen-binding fragment,and/or ADC therapeutic composition can be evaluated in a suitable animalmodel. For example, xenogeneic cancer models can be used, wherein cancerexplants or passaged xenograft tissues are introduced into immunecompromised animals, such as nude or SCID mice (Klein et al. (1997)Nature Med. 3:402-8). Efficacy may be predicted using assays thatmeasure inhibition of tumor formation, tumor regression or metastasis,and the like.

In vivo assays that evaluate the promotion of tumor death by mechanismssuch as apoptosis may also be used. In some embodiments, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

Further provided herein are methods of treating cancer. The antibodies,antigen-binding fragments, and/or ADCs disclosed herein can beadministered to a non-human mammal or human subject for therapeuticpurposes. The therapeutic methods entail administering to a mammalhaving a tumor a biologically effective amount of an antibody,antigen-binding fragment, and/or ADC comprising eribulin linked to atargeting antibody that binds to an antigen expressed, is accessible tobinding, or is localized on a cancer cell surface.

An exemplary embodiment is a method of delivering eribulin to a cellexpressing mesothelin, comprising conjugating eribulin to an antibodythat immune-specifically binds to a mesothelin epitope and exposing thecell to the antibody, antigen-binding fragment, and/or ADC. Exemplarytumor cells that express mesothelin for which the antibodies,antigen-binding fragments, and/or ADCs of the present disclosure areindicated include ovarian carcinoma cells, endometrioid cells, and lungsquamous cell mesothelioma cells.

Another exemplary embodiment is a method of reducing or inhibitinggrowth of a target antigen-expressing tumor (e.g., amesothelin-expressing tumor), comprising administering a therapeuticallyeffective amount of an antibody, antigen-binding fragment, and/or ADC.In some embodiments, the treatment is sufficient to reduce or inhibitthe growth of the patient's tumor, reduce the number or size ofmetastatic lesions, reduce tumor load, reduce primary tumor load, reduceinvasiveness, prolong survival time, and/or maintain or improve thequality of life. In some embodiments, the tumor is resistant orrefractory to treatment with the antibody or antigen-binding moiety ofthe ADC when administered alone, and/or the tumor is resistant orrefractory to treatment with eribulin when administered alone.

Moreover, antibodies of the present disclosure may be administered to anon-human mammal expressing mesothelin for veterinary purposes or as ananimal model of human disease. Regarding the latter, such animal modelsmay be useful for evaluating the therapeutic efficacy of the disclosedantibodies, antigen-binding fragments, and/or ADCs (e.g., testing ofdosages and time courses of administration).

Further provided herein are therapeutic uses of the disclosedantibodies, antigen-binding fragments, and/or ADCs. An exemplaryembodiment is the use of an antibody, antigen-binding fragment, and/orADC in the treatment of a target antigen-expressing cancer (e.g., amesothelin-expressing cancer) are also disclosed. Methods foridentifying subjects having cancers that express a target antigen (e.g.,mesothelin) are known in the art and may be used to identify suitablepatients for treatment with a disclosed antibody, antigen-bindingfragment, and/or ADC.

Another exemplary embodiment is the use of an antibody, antigen-bindingfragment, and/or ADC in a method of manufacturing a medicament for thetreatment of a target antigen-expressing cancer (e.g., amesothelin-expressing cancer).

The therapeutic compositions used in the practice of the foregoingmethods may be formulated into pharmaceutical compositions comprising apharmaceutically acceptable carrier suitable for the desired deliverymethod. An exemplary embodiment is a pharmaceutical compositioncomprising an antibody, antigen-binding fragment, and/or ADC of thepresent disclosure and a pharmaceutically acceptable carrier. Suitablecarriers include any material that, when combined with the therapeuticcomposition, retains the anti-tumor function of the therapeuticcomposition and is generally non-reactive with the patient's immunesystem.

Pharmaceutically acceptable carriers include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol, mesylate salt, and the like, aswell as combinations thereof. In many cases, isotonic agents areincluded, for example, sugars, polyalcohols such as mannitol, sorbitol,or sodium chloride in the composition. Pharmaceutically acceptablecarriers may further comprise minor amounts of auxiliary substances suchas wetting or emulsifying agents, preservatives or buffers, whichenhance the shelf life or effectiveness of the ADC.

Therapeutic formulations may be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like.Therapeutic protein preparations can be lyophilized and stored assterile powders, e.g., under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection. Therapeuticformulations may comprise an antibody, antigen-binding fragment, and/orADC or a pharmaceutically acceptable salt thereof, e.g., a mesylatesalt.

The antibodies, antigen-binding fragments, and/or ADCs disclosed hereinmay be administered at a dosage ranging from about 0.2 mg/kg to about 10mg/kg to a patient in need thereof. In some embodiments, an antibody,antigen-binding fragment, and/or ADC is administered to the patientdaily, bimonthly, or any time period in between. Dosages andadministration protocols for the treatment of cancers using theforegoing methods will vary with the method and the target cancer, andwill generally depend on a number of other factors appreciated in theart.

Various delivery systems are known and may be used to administer one ormore antibodies, antigen-binding fragments, and/or ADCs of the presentdisclosure. Methods of administering the antibodies, antigen-bindingfragments, and/or ADCs include, but are not limited to, parenteraladministration (e.g., intradermal, intramuscular, intraperitoneal,intravenous and subcutaneous), epidural administration, intratumoraladministration, and mucosal administration (e.g., intranasal and oralroutes). In addition, pulmonary administration may be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent. See, e.g., the compositions and methods for pulmonaryadministration described in U.S. Pat. Nos. 6,019,968, 5,985,320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and Intl. Publ. Nos. WO 1992/019244, WO 1997/032572, WO 1997/044013, WO1998/031346, and WO 1999/066903. The ADCs may be administered by anyconvenient route, for example, by infusion or bolus injection, or byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.). Administration can beeither systemic or local.

Therapeutic compositions disclosed herein may be sterile and stableunder the conditions of manufacture and storage. In some embodiments,one or more of the antibodies, antigen-binding fragments, and/or ADCs,or pharmaceutical compositions, is supplied as a dry sterilizedlyophilized powder or water free concentrate in a hermetically sealedcontainer and can be reconstituted (e.g., with water or saline) to theappropriate concentration for administration to a subject. In someembodiments, one or more of the prophylactic or therapeutic agents orpharmaceutical compositions is supplied as a dry sterile lyophilizedpowder in a hermetically sealed container at a unit dosage of at least 5mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, atleast 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg, or anyamount in between. In some embodiments, the lyophilized antibodies,antigen-binding fragments, and/or ADCs or pharmaceutical compositions isstored at between 2° C. and 8° C. in the original container. In someembodiments, one or more of the antibodies, antigen-binding fragments,and/or ADCs or pharmaceutical compositions described herein is suppliedin liquid form in a hermetically sealed container, e.g., a containerindicating the quantity and concentration of the agent. In someembodiments, the liquid form of the administered composition is suppliedin a hermetically sealed container of at least 0.25 mg/mL, at least 0.5mg/mL, at least 1 mg/mL, at least 2.5 mg/mL, at least 5 mg/mL, at least8 mg/mL, at least 10 mg/mL, at least 15 mg/mL, at least 25 mg/mL, atleast 50 mg/mL, at least 75 mg/mL, or at least 100 mg/mL ADC. The liquidform may be stored at between 2° C. and 8° C. in the original container.

In some embodiments, the disclosed antibodies, antigen-bindingfragments, and/or ADCs can be incorporated into a pharmaceuticalcomposition suitable for parenteral administration. The injectablesolution may be composed of either a liquid or lyophilized dosage formin a flint or amber vial, ampule, or pre-filled syringe, or other knowndelivery or storage device.

The compositions described herein may be in a variety of forms. Theseinclude, for example, liquid, semi-solid, and solid dosage forms, suchas liquid solutions (e.g., injectable and infusible solutions),dispersions or suspensions, tablets, pills, powders, liposomes, andsuppositories. The form depends on the intended mode of administrationand therapeutic application.

In various embodiments, treatment involves single bolus or repeatedadministration of the antibody, antigen-binding fragment, and/or ADCpreparation via an acceptable route of administration.

Patients may be evaluated for the levels of target antigen in a givensample (e.g., the levels of target antigen expressing cells) in order toassist in determining the most effective dosing regimen, etc. Anexemplary embodiment is a method of determining whether a patient willbe responsive to treatment with an antibody, antigen-binding fragment,and/or ADC of the present disclosure, comprising providing a biologicalsample from the patient and contacting the biological sample with theantibody, antigen-binding fragment, and/or ADC. Exemplary biologicalsamples include tissue or body fluid, such as an inflammatory exudate,blood, serum, bowel fluid, stool sample, or tumor biopsy (e.g., a tumorbiopsy derived from a patient having or at risk of a targetantigen-expressing cancer, e.g., a mesothelin-expressing cancer). Insome embodiments, a sample (e.g., a tissue and/or body fluid) can beobtained from a subject, and a suitable immunological method can be usedto detect and/or measure protein expression of the target antigen (e.g.,mesothelin). Such evaluations are also used for monitoring purposesthroughout therapy, and are useful to gauge therapeutic success incombination with the evaluation of other parameters.

In some embodiments, the efficacy of an antibody, antigen-bindingfragment, and/or ADC may be evaluated by contacting a tumor sample froma subject with the antibody, antigen-binding fragment, and/or ADC andevaluating tumor growth rate or volume. In some embodiments, when anantibody, antigen-binding fragment, and/or ADC has been determined to beeffective, it may be administered to the subject.

The above therapeutic approaches can be combined with any one of a widevariety of additional surgical, chemotherapy, or radiation therapyregimens. In some embodiments, the antibodies, antigen-bindingfragments, and/or ADCs or compositions disclosed herein areco-formulated and/or co-administered with one or more additionaltherapeutic agents, e.g., one or more chemotherapeutic agents.Non-limiting examples of chemotherapeutic agents include alkylatingagents, for example, nitrogen mustards, ethyleneimine compounds, andalkyl sulphonates; antimetabolites, for example, folic acid, purine orpyrimidine antagonists; anti-mitotic agents, for example, anti-tubulinagents such as eribulin or eribulin mesylate (Halaven™), vincaalkaloids, and auristatins; cytotoxic antibiotics; compounds that damageor interfere with DNA expression or replication, for example, DNA minorgroove binders; and growth factor receptor antagonists. In someembodiments, a chemotherapeutic agent may be a cytotoxic or cytostaticagent. Examples of cytotoxic agents include, but are not limited to,anti-mitotic agents, such as eribulin or eribulin mesylate (Halaven™),auristatins (e.g., monomethyl auristatin E (MMAE), monomethyl auristatinF (MMAF)), maytansinoids (e.g., maytansine), dolastatins, duostatins,cryptophycins, vinca alkaloids (e.g., vincristine, vinblastine),taxanes, taxols, and colchicines; anthracyclines (e.g., daunorubicin,doxorubicin, dihydroxyanthracindione); cytotoxic antibiotics (e.g.,mitomycins, actinomycins, duocarmycins (e.g., CC-1065), auromycins,duomycins, calicheamicins, endomycins, phenomycins); alkylating agents(e.g., cisplatin); intercalating agents (e.g., ethidium bromide);topoisomerase inhibitors (e.g., etoposide, tenoposide); radioisotopes,such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32,and radioactive isotopes of lutetium (e.g., Lu177); and toxins ofbacterial, fungal, plant or animal origin (e.g., ricin (e.g., ricinA-chain), diphtheria toxin, Pseudomonas exotoxin A (e.g., PE40),endotoxin, mitogellin, combrestatin, restrictocin, gelonin,alpha-sarcin, abrin (e.g., abrin A-chain), modeccin (e.g., modeccinA-chain), curicin, crotin, Sapaonaria officinalis inhibitor,glucocorticoid).

Also disclosed herein are uses of one or more of the disclosedantibodies, antigen-binding fragments, and/or ADCs in the manufacture ofa medicament for treating cancer, e.g., according to the methodsdescribed above. In some embodiments, the ADCs disclosed herein are usedfor treating cancer, e.g., according to the methods described above.

In various embodiments, kits for use in the laboratory and therapeuticapplications described herein are within the scope of the presentdisclosure. Such kits may comprise a carrier, package, or container thatis compartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) comprising one of theseparate elements to be used in a method disclosed herein, along with alabel or insert comprising instructions for use, such as a use describedherein. Kits may comprise a container comprising a drug moiety. Thepresent disclosure also provides one or more of the antibodies,antigen-binding fragments, and/or ADCs, or pharmaceutical compositionsthereof, packaged in a hermetically sealed container, such as an ampouleor sachette, indicating the quantity of the agent.

Kits may comprise the container described above and one or more othercontainers associated therewith that comprise materials desirable from acommercial and user standpoint, including buffers, diluents, filters,needles, syringes; carrier, package, container, vial and/or tube labelslisting contents and/or instructions for use, and package inserts withinstructions for use.

A label may be present on or with the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, such as a prognostic, prophylactic, diagnostic, orlaboratory application. A label may also indicate directions for eitherin vivo or in vitro use, such as those described herein. Directions andor other information may also be included on an insert(s) or label(s),which is included with or on the kit. The label may be on or associatedwith the container. A label may be on a container when letters, numbers,or other characters forming the label are molded or etched into thecontainer itself. A label may be associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. The label may indicate that the compositionis used for diagnosing or treating a condition, such as a cancer adescribed herein.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the invention in detail, the samewill be more clearly understood by reference to the following examples,which are included for purposes of illustration only and are notintended to be limiting.

EXAMPLES Example 1: Chimeric Antibody Generation Against HumanMesothelin

Chimeric antibodies containing rabbit and human immunoglobulin sequenceswere generated according to the procedures described below. Antibodieswere analyzed for binding to human mesothelin and epitope binding.Initial ADC cytotoxicity of the recombinant chimeric anti-mesothelinantibodies was evaluated in human cell lines expressing varying levelsof mesothelin. Lead antibodies for humanization and ADC development aredescribed in Examples 2-3.

1.1 Reagents and Materials

1.1.1 Antibodies

The antibodies used in the following studies are the rabbit-humanchimeric (−xi) form of anti-human mesothelin antibodies and have anunpaired cysteine at light chain position 80 (LCcys80). The antibodieswere purified and decysteinylated as described below in Section 1.5.Final protein content was assessed by BCA assay and SDS-PAGE.

1.1.2 Conjugatable Cytotoxins and LCcys80 ADCs

Linker-cytotoxin compounds used in the following studies includeMal-PEG₂-Auristatin F. The antibodies were conjugated withMal-PEG₂-Auristatin F at a molar ratio of 1:5 (mAb:payload). ConjugatedLCcys80 antibodies were purified using desalting chromatography with 2×5mL HiTrap desalting columns (GE Healthcare) on an AKTA FPLC, using1×DPBS as running buffer. Final protein content was determined by BCAassay.

1.1.3 Tumor Cell Lines

Human tumor cell lines used in the analyses of rabbit-human chimericADCs included A431-K5 (human melanoma cells A431 stably transfected withhuman mesothelin, MSLN^(hi)), A431 (MSLN^(lo)), and OVCAR3 (humanovarian carcinoma, MSLN^(hi)). A431-K5 cells were obtained from theNational Cancer Institute. Cell lines used were obtained directly fromthe American Type Culture Collection (ATCC).

1.1.4 Other Reagents

All reagents used were obtained from commercial suppliers atresearch-grade or higher, unless otherwise indicated.

1.2 Generation of Antibodies in Rabbits Against Human Mesothelin

Human mesothelin cDNA from vector p0301 was cloned into an Aldevronexpression vector (pB8-Mesothelin-hum). Two rabbits were then immunizedwith the immunization vector pB8-mesothelin-human. After four geneticapplications, immune sera were taken at day 52 of the immunizationprotocol. Rabbit immune sera was diluted 1:1000 or 1:5000 in PBScontaining 1% BSA, and was tested by flow cytometry using mammaliancells previously transiently transfected with the human mesothelin cDNAcloned into an Aldevron expression vector (pB1-mesothelin-hum) andmammalian cells transiently transfected with an irrelevant cDNA clonedinto the same vector. Antibodies from the immune sera were then detectedwith 10 μg/mL goat anti-rabbit IgG R-phycoerythrin (Southern Biotech,#4030-09). Immunization, flow cytometry, and cryo-conserving cells wereperformed by Aldevron (Dreiburg, Germany).

1.3 High Throughput Screening of Cultures Producing Anti-MesothelinAntibodies

1.3.1 Cell Culture

Cryo-conserved rabbit lymph node cells (2.0×10⁷ cells) were thawed thenactivated with 2.5 μg/mL of lectin from Phytolacca americana andrecovered with DNAse I for one hour at 37° C. with 5% CO₂. The cellswere seeded at 5 cells per well on a 384 well plate with feeder cells(CHOs expressing rabbit CD154) and cultured in complete IMDM (IMDMsupplemented with 10% FBS, 2 mM L-glutamine, 1×MEM NEAA, 1 mM SodiumPyruvate, 50 U/mL Penicillin, 50 μg/mL Streptomycin, 55 μM 2-Me) thatcontained 10.5 ng/mL human IL2 and 10.5 ng/mL human IL21 cytokines(PeproTech).

1.3.2 Isolation of Rabbit IgG and Polyclonal Antibodies Against HumanMesothelin

On week 2, the wells producing rabbit IgG antibody were identified byIgG FRET using europium cryptate. Wells producing IgG were screened forthe presence of rabbit IgG Fcγ antibody by ELISA against plates coatedwith 1 μg/mL of CHO-MT40 mesothelin. The cultures producing mesothelinspecific rabbit IgG were confirmed by ELISA screening against 1 μg/mL ofmesothelin and counter screened against 1 μg/mL of CD73-his. FRET andELISA were performed on the Biomek® FX robotic system (Beckman).

1.3.3 mRNA Gene Rescue of Rabbit Antibodies Against Human Mesothelin

Total RNA was isolated from wells producing rabbit IgG anti-mesothelinantibodies using RNAqueous™-96 Total RNA Isolation Kit (Ambion). cDNAwas synthesized and light and heavy chain variable regions wereamplified by PCR using Platinum Taq one step RT-PCR kit (Invitrogen)using in-house primers (Table 11). The light and heavy chain variableregions were amplified with nested primers (Table 12) using Platinum TaqAmplification Kit and a thermocycler (40 cycles, 1 min 94° C., 1 min 54°C., 1.5 min 68° C.). Amplified DNA template was visualized by gelelectrophoresis, purified by QIAquick 96 PCR Purification Kit (Qiagen)and DNA sequence was determined by GeneWiz (South Plainfield, N.J.)using in-house primers (Table 13). DNA Sequences were analyzed againstV-gene and J-gene rabbit families (IMGT/V-QUEST) and against in-houseIn-Fusion primer database (Blastn). In-Fusion primers (Table 14) wereeither identified or designed containing a human Fc linker added to the5′ end for the V and J gene primer. Primers were synthesized by IDT(Coralville, Iowa).

TABLE 11 Primer sequences used for one step RT-PCR Gene 5′ Primer3′ Primer Heavy TYCTCCTGGTCRCTSYGCTC TTGGTGTTGGTGGCTGGGTG(SEQ ID NO: 37) (SEQ ID NO: 38) Light GGGCCCCCACTCAGCTGCTGGTTBTACTGKTMTYGATGCC (SEQ ID NO: 39) (SEQ ID NO: 40)

TABLE 12 Primer sequences used for PCR Gene 5′ Primer 3′ Primer HeavyTYCTCCTGGTCRCTSYGCTC TTGGTGTTGGTGGCTGGGTG (SEQ ID NO: 41)(SEQ ID NO: 42) Light ACTCAGCTGCTGGGGCTCCT GTTBTACTGKTMTYGATGCC(SEQ ID NO: 43) (SEQ ID NO: 44)

TABLE 13 Primer sequences used for DNA template sequencing Gene3′ Primer Heavy TTGGTGTTGGTGGCTGGGTG (SEQ ID NO: 45) LightGTTBTACTGKTMTYGATGCC (SEQ ID NO: 46)

TABLE 14 Primer sequences used for In-Fusion PCR for sample 345A12 Gene5′ Primer 3′ Primer Heavy gccaccggcgtgcactccCAG gggcccttggtggatgcTGARTCGYTGGAGGAGTCCGGGGG GAGACRGTGACSAGGGTSCC (SEQ ID NO: 47)(SEQ ID NO: 48) Light gccaccggcgtgcactccGCC agccacagttcgTTTGACSACTATGATATGACCCAGACTCCA CACCTCGGTCCC (SEQ ID NO: 49) (SEQ ID NO: 50)1.3.4 PCR Fragments

PCR-amplified variable domains included 15 base-pairs at the 5′ and 3′ends homologous to the cloning site within the subcloning vector. PCRfragments were subcloned into an expression plasmid containing a humangamma (p1974 pC+751Z-ldr-InFusion-hugamma) or kappa constant region(p1975 pC+75IB-ldr-InFusion-hukappa) using an In-Fusion HD cloning kit(Clontech) according to the manufacturer's protocol. 1 μL of theIn-Fusion reaction was transformed into Stellar Competent Cells(Clontech) according to manufacturer's protocol. Transformants weregrown in 1 mL TB medium (Teknova) overnight at 37° C. on a microtiterplate shaker. The next day, cultures were miniprepped with a QIAprep 96Turbo miniprep kit (Qiagen) using an epMotion 5075 according to themanufacturer's protocol.

1.3.5 Gene Synthesis Fragments

Humanized heavy and light variable domains were codon-optimized forexpression in Chinese hamster ovary (CHO) cells and were synthesized byGeneArt. The variable domains were synthesized with a Kozak translationinitiation sequence and an Ig secretion leader sequence, and included 15base-pairs at the 5′ and 3′ ends homologous to the cloning site withinthe subcloning vector. PCR fragments synthesized by GeneArt weresubcloned into an expression plasmid containing a human gamma (p1974pC+75IZ-ldr-InFusion-hugamma) or kappa constant region (p1975pC+75IB-ldr-InFusion-hukappa) using an InFusion HD cloning kit(Clontech). All clones were sequenced to confirm the presence andfidelity of the inserts.

1.4 Transient mAb Production

1.4.1 HEK Cells

For each milliliter of 3×10⁶ cells to be transfected with ExpiFectamine(ThermoFisher), 333.3 ng HC plasmid and 333.3 ng LC plasmid wereincubated for 5-10 min in 50 μL Opti-MEM (ThermoFisher). Likewise, 2.67μL ExpiFectamine was incubated in 50 μL Opti-MEM. The ExpiFectaminesolution was added to the DNA mixture, and incubated for 20-30 min atroom temperature. The DNA:ExpiFectamine mixture was added to the cellswhile swirling and incubated at 37° C., 8% CO₂, shaking at 125 rpm. Thefollowing day, 5 μL of enhancer 1 and 50 μL of enhancer 2 per mL ofcells were added to the transfection with continued incubation foranother 7-10 days. After 48-72 hours, cells were fed at a finalconcentration of 10 g/L Yeastolate (BD Biosciences), 5 mM valeric acid(Sigma-Aldrich), and 1:100 CD Lipid Concentrate (ThermoFisher).

1.4.2 CHO Cells

For each milliliter of 6×10⁶ cells to be transfected with ExpiFectamineCHO (ThermoFisher), 500 ng HC plasmid and 500 ng LC plasmid were mixedin Opti-PRO (ThermoFisher) in 40 μL total volume. Likewise, 3.2 μLExpiFectamine CHO was mixed in 36.8 μL Opti-PRO. The ExpiFectamine CHOsolution was added to the DNA mixture, and incubated for 1-5 min at roomtemperature. The DNA:ExpiFectamine CHO mixture was added to the cellswhile swirling and incubated at 37° C., 8% CO₂, shaking at 125 rpm. Thefollowing day, 6 μL of enhancer and 160 μL of feed per mL of cells wereadded to the transfection, and cells were transferred to 32° C., 5% CO₂.At day 5, an additional 160 μL of feed per mL of cells was added. Atdays 12 to 14, the supernatants were harvested.

1.5 mAb Purification and Decysteinylation

1.5.1 Antibody Purification

Prosep-vA High Capacity Protein A resin (Millipore) was equilibratedwith DPBS, and 50 μL were added to 2 mL of sample. Following incubationat room temperature for 1 hour, the medium and resin were added to afilter plate and washed twice with 1 mL DPBS. The sample was eluted fromthe resin by addition of 400 μL 0.1 M Glycine, pH 2.9 followed bycentrifugation at 15,000×g for 30 sec. The sample was neutralized with20 μL of 1 M Tris, pH 8.0. The samples were concentrated toapproximately 100 μL by centrifugation at 15,000×g for 5 min using 0.5mL Amicon Ultra, 10 k cutoff filters (Millipore) and werebuffer-exchanged into DPBS using 0.5 mL Zeba desalting columns, 7K MWCO,according to the manufacturer's protocol. mAb concentration wasdetermined by measuring AU280 and converted to mg/mL using the mAb'sextinction coefficient.

1.5.2 Cysteine Decapping

Purification was performed using an AKTA Xpress purification platform(GE Healthcare). Up to 1 L of conditioned medium was loaded onto a 5 mLMabSelect column (GE Healthcare) equilibrated in 20 mM sodium phosphate,150 mM NaCl, pH 7.0. The column was washed extensively withequilibration buffer following loading until a stable baseline wasobserved. Bound material was eluted using 100 mM glycine, pH 2.9. Elutedmaterial was immediately injected on to a 26/10 HiPrep desalting column(GE Healthcare) equilibrated in 1× phosphate-buffered saline (PBS) andeluted in the same buffer. Peak fractions were pooled. Material wasanalyzed for protein content by BCA assay (ThermoFisher) andelectrophoresis by reducing and non-reducing SDS-PAGE.

1.6 Initial Screening and Characterization of Recombinant ChimericAnti-Mesothelin Antibodies for ADC Development

Anti-mesothelin antibodies were transiently expressed and cultured in 96deep-well plates using Expi-293 medium. Antibodies from the supernatantwere purified and decysteinylated as described above. The antibodieswere conjugated using Mal-PEG₂-Auristatin F as payload using a molarratio of 1:5 (mAb:payload). Conjugated antibodies were desalted toremove extra free payload using Thermo Zeba spin desalting plates.

1.7 Binding Characterization

1.7.1 Anti-Mesothelin Epitope Binning Using Octet

The antibody binding epitope to mesothelin was initially characterizedusing Octet, with Streptavidin tip, using a customized binding assay.Epitope binding of the anti-mesothelin antibodies were normalized to theepitope bound by a known anti-mesothelin antibody, MORAb-009(Amatuximab). Antibodies were grouped based on their binding to thesame, nearby, or different epitope as MORAb-009. Steps were repeateduntil all antibodies aligned with different epitope binning. Bindingaffinity was ranked as high, medium and low based on the Octet results.All binding steps were conducted in the PBST buffer containing 0.2% BSA.

1.7.2 Surface Plasmon Resonance (BIAcore) Binding Analysis

Anti-mesothelin antibody binding affinity to mesothelin was measured byBIAcore (BIAcore T-100, GE healthcare, #1426075), using a series S CMSchip. Antibody concentrations were adjusted to 1 μg/mL and mesothelin(50 μg) to 100 nM in 1×HBS-P+ buffer (GE Healthcare). Anti-humanantibody capture chip was prepared according to the manufacturer'sprotocol using a CMS chip with immobilization wizard. Final captureantibody levels were 8000-9000 RU, in HBS-P+. Chip was prepared forassay with five cycles of 300 sec buffer injection followed by 30 secregeneration, all at 30 μL/min across all four flow cells. Antibodieswere captured on flow cells 2-4 by sequential injections of individualligand solutions for 90 sec at 10 μL/min. Analyte injection was done ina single-cycle kinetics manner by sequential injections of analytesolutions from low to high concentration for 240 sec each at 30 μL/min.Detection was 2-1, 3-1, 4-1. Double-referencing was performed by asequence of identical ligand capture injections, followed by 5buffer-only injections for 240 sec each, dissociation for 1800 sec, andregeneration as above. All ligands were analyzed for binding tomesothelin in duplicate. Kinetic analysis was performed usingBIAEvaluations software using a 1:1 Langmuir fitting model. On-rate,off-rate, and affinity constants were averaged from duplicate runs.

1.8 In Vitro Cytotoxicity Analysis

A431, A431-K5, and OVCAR3 cells were sub-cultured and seeded at 5,000cells/well in complete growth medium in 96-well tissue culture plates,and incubated at 37° C., 5% CO₂ overnight (16 hours). Test reagents wereserially diluted 1:3 in 2 mL deep-well dilution plates, starting at 200nM (10 dilutions total). Diluted samples (100 μL) were added to the cellplates (starting concentration of test samples at 100 nM). Plates wereincubated at 37° C., 5% CO₂ for an additional 5 days. Medium was thendiscarded, and plates were washed once with 200 μL DPBS, stained with 50μL of 0.2% Crystal Violet solution at room temperature for 15 min, andthen washed extensively with tap water. Plates were air-dried, andCrystal Violet was dissolved with 200 μL of 1% SDS solution. Plates wereread at 570 nm. Data was analyzed using GraphPad Prism 6.

1.9 Results

1.9.1 Rabbit Immunization

Two rabbits were DNA immunized with the plasmid pB8-mesothelin-human forfour genetic applications. Immune sera were taken at day 52 of theimmunization protocol, diluted 1:1000 or 1:5000 in PBS containing 1%BSA, and was tested by flow cytometry using mesothelin-expressing cells.Sera from both immunized rabbits bound the mesothelin-expressing cells,which were cells transfected with pB1-mesothelin-hu (FIG. 1 , lowercurves). Conversely, sera from immunized rabbits did not bind cellstransfected with an irrelevant cDNA (FIG. 1 , upper curves).

1.9.2 High Throughput Screening of Cultures Producing Rabbit PolyclonalAntibodies Against Human Mesothelin

Rabbit lymph node cells were harvested and cryo-preserved. Cells (2×10⁷cells) from thawed lymph node were seeded at 5 cells per well on a 384well plate with feeder cells and cultured in complete IMDM containing10.5 ng/mL human IL-2 and 10.5 ng/mL human IL-21 cytokines. Wellsproducing rabbit IgG antibody were identified two weeks followingseeding via IgG FRET using europium cryptate and 18,715 IgG-producingcultures were screened by ELISA for human mesothelin reactivity.Eighty-five mesothelin-specific cultures were re-confirmed forreactivity to mesothelin and counter-screened against reactivity tohuman CD73. There were 54 confirmed cultures producing rabbit Fcγantibody that bound mesothelin above 0.2 OD₄₅₀ with no cross reactivityto CD73 (FIG. 2 ). Primary ELISA results are shown by the right-most setof bars, secondary ELISA results are shown by the left-most set of bars,and human CD73 binding is shown by the middle set of bars.

1.9.3 RT-PCR, Sequencing, and Cloning of Variable Regions

Total RNA was isolated from 54 confirmed cultures producing rabbit IgGanti-mesothelin antibodies, cDNA was synthesized by RT-PCR, and lightand heavy chain variable regions were PCR amplified. Fifty-two DNAsequences were analyzed using V-gene and J-gene rabbit families(IMGT/V-QUEST) and 51 were PCR amplified with primers specific forIn-Fusion cloning into constant region expression vectors (FIG. 3 ). Atotal of 48 antibodies were cloned into human constant region expressionvectors and were subsequently transfected into expi293F cells. Antibodywas detected in 45 of the 51 transfectants (FIG. 4 ), rabbit variableregions were In-Fusion cloned into human constant region expressionvectors.

1.9.4 Initial Screening of ADCs Against Mesothelin-Expressing Cells

Chimeric rabbit anti-human mesothelin (rb-hu-xi anti-MSLN) antibodieswere purified according to method described in Section 1.5. The proteinconcentration of purified antibodies were determined (FIG. 5 ). Tocomplete the screening of anti-mesothelin antibody for ADC development,micro-conjugation of anti-mesothelin antibody with Mal-PEG₂ Auristatin Fwas performed, and ADCs were characterized in the in-vitro cell basedpotency assay using OVCAR3, A431-K5 and A431 cell lines, where OVCAR3and A431-K5 expressed high level of mesothelin, and A431(MSLN⁻) was usedas control cell line for evaluating off-target killing and specificityof ADCs (FIG. 6 ).

1.9.5 Epitope Binding of Anti-Mesothelin Antibodies

The binding epitope to mesothelin of the 48 anti-mesothelin antibodieswere characterized using Octet as indicated in Section 1.7.1. Sixdifferent epitopes were identified for the antibodies, and 102A6observed no binding in the current format by Octet (FIG. 7 ). Antibodybinding affinity to mesothelin was measured by BIAcore, as indicated inSection 1.7.2. The binding affinity results are summarized in Table 15.

TABLE 15 Anti-mesothelin antibody binding affinity to mesothelin EpitopeBin Curve k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) Rmax (RU) Chi² (RU²) M09-31.95E+06 2.92E−04 1.50E−10 40.19 0.646 55B4-1 1.67E+06 8.32E−03 4.98E−0942.33 1.72 55B4-2 1.04E+06 5.52E−03 5.33E−09 42.6 0.586 MORAb-00962B10-1 2.10E+06 8.57E−03 4.08E−09 47.6 3.54 62B10-2 9.91E+05 6.71E−036.77E−09 46.58 0.398 131F9-1 8.36E+05 7.23E−03 8.65E−09 48.89 0.458131F9-2 8.71E+05 6.79E−03 7.79E−09 48.71 0.639 145D4-1 8.05E+05 6.83E−038.48E−09 50.61 0.489 145D4-2 8.69E+05 6.90E−03 7.95E−09 51.21 0.681342P20-1 1.74E+06 2.27E−04 1.30E−10 29.15 0.498 342P20-2 2.32E+062.51E−04 1.08E−10 27.51 0.447 Xi-33O11 1.99E+06 6.68E−04 3.35E−10 35.890.169 43F8-1 7.10E+05 6.37E−03 8.97E−09 35.29 0.48 #1 43F8-2 7.77E+055.82E−03 7.48E−09 36.43 0.78 201C15-1 2.74E+05 1.29E−04 4.70E−10 63.950.75 201C15-2 2.64E+06 3.45E−04 1.31E−10 58.09 0.568 X-237N18 1.27E+064.28E−03 3.38E−09 41.45 0.295 Xi-393L14 1.10E+06 2.57E−04 2.33E−10 46.470.627 #3 Xi-383I18(AuF) 1.01E+06 2.81E−04 2.80E−10 43.9 0.494 346C6-16.82E+05 4.81E−04 7.06E−10 44.85 0.218 346C6-2 1.15E+06 6.09E−045.29E−10 45.06 0.503 345A12-1 2.89E+06 4.12E−04 1.43E−10 39 0.793 #5345A12-2 2.85E+06 3.89E−04 1.36E−10 38.63 0.833 120N18-1 6.26E+055.26E−04 8.41E−10 50.28 0.323 120N18-2 6.57E+05 5.13E−04 7.80E−10 50.890.395 82M2-1 7.18E+05 4.35E−04 6.06E−10 47.12 0.493 #6 82M2-2 8.98E+054.74E−04 5.28E−10 48.64 0.961 M09/#2 264E24-1 4.01E+05 5.81E−05 1.45E−1048.8 0.347 hybrid 264E24-2 4.10E+05 5.47E−05 1.34E−10 49.33 0.423 M09/#3238B22-1 2.60E+05 3.03E−03 1.17E−08 153.1 6.95 hybrid 238B22-2 4.31E+056.03E−03 1.40E−08 57.04 0.318

Based on the results above, fifteen antibodies that cover all epitopebins were selected for scale-up conjugation and characterization, asindicated in Table 16. 102A6A was also selected based on favorablein-vitro potency when conjugated to auristatin F.

TABLE 16 Fifteen selected anti-mesothelin antibodies and their epitopebins Epitope Bin Lead Antibodies Bin #1 33O11, 210C15 Bin #2 111B10,324O5, 178F16, 264E24 Bin #3 237N18, 383I18, 393L14, 346C6 Bin #4 62B10,55B4, MORAb009 Bin #5 120N18, 345A12 No binding 102A6A

Example 2: Humanization of Anti-Mesothelin ADCs

Humanized anti-mesothelin antibodies were generated according to theprocedures described below. Antibodies and ADCs were analyzed forretained binding activity to human mesothelin and cell killing potencytoward cells expressing mesothelin. Antibodies were also biophysicallycharacterized for drug loading, aggregation, thermal stability, andserum and matrix stability. Lead humanized antibodies and ADCs wereevaluated in vivo, as described in Example 3.

2.1 Reagents and Materials

2.1.1 Antibodies

The antibodies used in the following studies have an unpaired cysteineat light chain position 80 (LCcys80) and include both the rabbit-humanchimeric (−xi) and humanized (−zu) forms of the anti-human mesothelinantibodies 33O11, 201C15, 111B10, 324O5, 178F16, 264E24, 237N18, 383I18,393L14, 346C6, 62B10, 55B4, MORAb009, 120N18, 345A12, and 102A6A2. Theantibodies were batch purified using a Prosep-vA High Capacity Protein Aresin and Zeba desalting columns. Conditioned medium was purified anddecysteinylated as described in Section 1.5 (Example 1). Final proteincontent was assessed via BCA assay and SDS-PAGE.

2.1.2 Conjugatable Cytotoxins and LCcys80 ADCs

Linker-cytotoxin compounds used in the following studies includemaleimide-VCP-eribulin, maleimide-VCP-cryptophycin, andmaleimide-VCP-eribulin dimer. Conjugated antibodies were purified usingdesalting chromatography with HiTrap desalting columns (GE Healthcare)equilibrated in 1×DPBS. Final protein content was determined by BCAassay.

2.1.3 Tumor Cell Lines

Human tumor cell lines used in the analyses of rabbit-human chimericADCs included A431 (human melanoma cells, MSLN^(neg)), A3 (A431 stablytransfected with human mesothelin, MSLN^(hi)) OVCAR3 (human ovariancarcinoma cells, MSLN^(hi)), HEC-251 (human endometrioid, MSLN^(med)),and H226 (human lung squamous cell mesothelioma, MSLN^(lo)). All celllines used were obtained directly from the American Type CultureCollection (ATCC), with the exception of A3, which was generated atMorphotek from the A431 parental cell line and HEC-251, which wasobtained from JCRB.

2.1.4 Other Reagents

All reagents used were obtained from commercial suppliers atresearch-grade or higher, unless otherwise indicated.

2.2 Biophysical Characterization of ADCs

2.2.1 SEC-HPLC Aggregation Analysis

SEC-HPLC analysis was conducted using Agilent 1200 HPLC system.AdvanceBio SEC 300A (2.7 μm, 7.8×50 mm, serial no. 0006344424-13, batchno. 0006344424) guard column was connected to AdvanceBio SEC 300Aanalytical column (2.7 μm, 7.8×300 mm, serial no. 0006336837-4, batchno. 0006336837), equilibrated in 0.1 M sodium phosphate, 0.15 M sodiumchloride, 5% IPA, pH 7.4, at flow rate of 0.5 mL/min.

Aggregation of LCcys80 ADCs was analyzed by size-exclusion,high-performance liquid chromatography (SEC-HPLC) using an Agilent 1200HPLC. Antibodies and ADCs were prepared at 2 mg/mL in 1×DPBS, 8 μL (16μg) of each sample was injected and run for 36 min. All data wereanalyzed using Agilent ChemStation software. Percent aggregation,percent monomer, and percent fragmentation were reported.

2.2.2 Hydrophobic Interaction Chromatography (HIC-HPLC) DAR Analysis

DAR was analyzed using hydrophobic interaction chromatography (HIC-HPLC)on an Agilent HPLC 1260 system. Samples were injected onto a TSKgelEthyl-5PW column (TOSOH Bioscience, 7.5 mm ID×7.5 cm, 10 μm, nonporoussize), and eluted from the column with a 3 min equilibration in 100% ofmobile phase A, a 15 min gradient (0-100% B), a 5 min hold in 100% B, a1 min change to 100% A, and a 5 min re-equilibration in 100% of mobilephase A, at 0.7 mL/min. Mobile phase A was 25 mM sodium phosphate, 1.5 Mammonium sulfate, pH 7.0. Mobile phase B was 25 mM sodium phosphate, 25%isopropanol, pH 7.0. Detection was performed at 280 nm (reference 320nm). DAR was determined by the formula:[AUC+1+2(AUC+2)+3(AUC+3)+ . . . n(AUC+n)]/ΣAUCtot]where AUC+1 is the area under the curve for the antibody peakcorresponding to ADC conjugated with one cytotoxin, and AUC+2 is thearea under the curve for the antibody peak corresponding to ADCconjugated with two cytotoxins. ΣAUCtot is the combined area under thecurve for the conjugated and unconjugated peaks (DAR=0, 1, and 2).2.2.3 Liquid Chromatography/Mass Spectrometry (LC-MS) DAR Analysis

DAR was also analyzed using an LC-MS method with a Waters Alliance HPLCwith SQD/PDA detection. Samples were injected onto a Proteomix RP-1000column (5 μm, 1000 Å, 4.6 mm×15 cm, Sepax) at 65° C., and eluted with a3 min equilibration in 25% B, a 27 min linear gradient from 25%-55% B, a5 min hold in 55% B, a 1 min change to 90% B, a 5 min hold at 90% B, a 1min change back to 25% B, and a 5 min re-equilibration at 25% B. Mobilephase A was 0.1% TFA in water, and mobile phase B was 0.1% TFA inacetonitrile. Elute was then 10:1 split into PDA and SQD detectors. SQDdetector was set up as ES positive, capillary voltage at 3.5 KV, conevoltage at 50 V, extractor at 5 V, and RF lens at 0.3 V, sourcetemperature at 150° C., desolvation temperature at 350° C. Mass data wasacquired at 200-2000 m/z for 40 min, continuum mode, and scan time 1sec. Data was analyzed and deconvoluted offline using MassLynx andMaxEnt1. DAR was calculated using the formula:2[[AUCLC+1+2(AUCLC+2)+3(AUCLC+3)+ . . .n(AUCLC+n)]/ΣILCtot]+2[[AUCHC+1+2(AUCHC+2)+3(AUCHC+3)+ . . .n(AUCHC+n)]/ΣAUCHCtot]where AUCLC+1 is area under the curve of the light chain peak conjugatedwith one cytotoxin, AUCLC+2 is area under the curve of the light chainpeak conjugated with two cytotoxins, etc. AUCHC are the area under thecurve of the corresponding heavy chains, and ΣAUCLCtot and ΣAUCHCtot arethe combined area under the curve of all unconjugated and conjugatedlight chains and heavy chains, respectively.2.3 Binding Characterization2.3.1 Anti-Mesothelin Epitope Binning Using Octet

The antibody binding epitope to mesothelin was initially characterizedusing Octet, with Streptavidin tip, using a customized binding assay.Epitope binding of the anti-mesothelin antibodies were normalized to theepitope bound by a known anti-mesothelin antibody, MORAb-009. Antibodieswere grouped based on their binding to the same, nearby, or differentepitope as MORAb-009. Steps were repeated until all antibodies alignedwith different epitope binning. Binding affinity was ranked as high,medium and low based on the octet results. All binding steps wereconducted in the PBST buffer containing 0.2% BSA.

2.3.2 Surface Plasmon Resonance (BIAcore) Binding Analysis

Anti-mesothelin antibody binding affinity to mesothelin was measured byBIAcore (BIAcore T-100, GE healthcare, #1426075), using a series S CMSchip. Antibody concentrations were adjusted to 1 μg/mL and mesothelin(50 μg) to 100 nM in 1×HBS-P+ buffer (GE Healthcare). Anti-humanantibody capture chip was prepared according to the manufacturer'sprotocol using a CMS chip with immobilization wizard. Final captureantibody levels were 8000-9000 RU, in HBS-P+. Chip was prepared forassay with five cycles of 300 sec buffer injection followed by 30 secregeneration, all at 30 μL/min across all four flow cells. Antibodieswere captured on flow cells 2-4 by sequential injections of individualligand solutions for 90 sec at 10 μL/min. Analyte injection was done ina single-cycle kinetics manner by sequential injections of analytesolutions from low to high concentration for 240 sec each at 30 μL/min.Detection was 2-1, 3-1, 4-1. Double-referencing was performed by asequence of identical ligand capture injections, followed by 5buffer-only injections for 240 sec each, dissociation for 1800 sec, andregeneration as above. All ligands were analyzed for binding tomesothelin in duplicate. Kinetic analysis was performed usingBIAEvaluations software using a 1:1 Langmuir fitting model. On-rate,off-rate, and affinity constants were averaged from duplicate runs.

2.4 Differential Scanning Calorimetry (DSC) Thermal Stability Analysis

VP Capillary Differential Scanning Calorimeter (VP-CapDSC; Microcal,VP-CapDSC, #12-07-149 with Origin-7 graphing and MicroCal VP-CapillaryDSC Software v.2.0) was used to decipher and compare the higher orderstructure and thermal stability of various F(ab′)2 fragments andcontrols. Samples were prepared on 96-well assay plates (MicroliterAnalytical Supply) using 20% Contrad solution and analyzed inauto-sampler at 10° C.

2.5 Capillary Isoelectric Focusing (cIEF) Analysis

Auto-sampler reagents were filled according to the CFR installation andstart-up procedures. Hemoglobin was used as system stability standard.Default settings for batch data analysis was used. Focus period #1 wasperformed for 1 min at 1,500 V for both the system suitability standardsand samples. Focus period #2 was performed for 5 min at 3,000 V for thesystem suitability standards and 11 min at 3,000 V for the TIGC samplesand matched buffer controls. Duplicate TIGC samples used Focus period #2at 4.5 min and all samples were bracketed with a system suitabilitystandard. Samples were automatically integrated using a peak widthparameter of 0.1 and threshold of 5 and integrated between pI 7.5-9.4.

2.6 Preparation of DAR2 and DAR6 MORAb-109 ADCs

The 345A12-HC15-LC4 CHOZN cell line was cultured in a wave bag (20 L)until viability <30% and concentrated to 2 L using TFF. Antibody wascaptured on Amosphere A3 resin preequilibrated in 20 mM sodiumphosphate, 10 mM EDTA, pH 7.2, washed in the same buffer until a stablebaseline was achieved (to remove unbound material), then reducedon-column for 8 hours using 20 mM sodium phosphate, 10 mM EDTA, 10 mMcysteine, pH 7.2 at low flow rate, then re-oxidized on-column for 60hours using 20 mM Tris, pH 7.5. Bound material was eluted in 0.1 Mglycine, pH 3.0, then diafiltered into 1×PBS, 2 mM EDTA, pH 7.4 andconcentrated to >10 mg/mL. Final recovery was 100%.

For DAR2 MORAb-109, maleimide-VCP-eribulin was added (in DMSO) at amolar ratio of 1:2.5 (mAb:payload) for 1 hour at room temperature.Following conjugation, material was diluted to 2 mg/mL, diafiltered into1×PBS, 2 mM EDTA to remove unconjugated linker-payload and concentratedto 5 mg/mL. DAR2 material was purified by preparative Ether-5PW HICchromatography. Final material was characterized by SEC-HPLC, RP-HPLC,and HIC-HPLC.

For DAR6 MORAb-109, purified/decysteinylated antibody was diluted to 7.5mg/mL in 1×PBS, 2 mM EDTA and further reduced by adding an equal volumeof 250 μM TCEP in the same buffer for 50 min, then an equal total volumeof 50% Propylene glycol in 1×DPBS/1 mM EDTA was added, then finallymaleimide-VCP-eribulin at molar ratio of 1:8 (mAb:payload), incubated atroom temperature for 1 hour. ADC was purified by G-25 chromatography toremove unconjugated payload and formulated into 1×PBS, 2 mM EDTA. Finalmaterial was characterized by SEC-HPLC, RP-HPLC, and HIC-HPLC.

2.7 In Vitro Serum Stability

Anti-mesothelin ADCs (maleimide-VCP-eribulin as payload) were preparedat 0.5 mg/mL either in PBS or human serum. Samples were incubated at 37°C. for 0, 24, 48, 72, 96, or 240 hours, then transferred to −80° C. forstorage. All samples thawed to ambient temperature, and single dilutionof 1:2,000 for testing. Samples were tested for total mAb, total ADC,and in cell based potency. Total mAb assay was developed as stepwisesandwich format on Gyrolab XP, captured with biotinylated mesothelin,and detected with Alexa Fluor 647 anti-IgG1 Fc. Quantifiable ranges forthe total mAb and the intact ADC assays were 6.25-800 ng/mL and 6.25-800ng/mL, respectively. Standard curve and QCs were made with MORAb-109(345A12-HC15-LC4-VCP-eribulin).

2.8 In Vitro DAR-Sensitive Matrix Stability of MORAb-109 ADCs

MORAb-109 (345A12-HC15-LC4-VCP-eribulin) DAR 2 was prepared at 0.1 mg/mLin either PBS or human, monkey, rat, or mouse serum in triplicate.Samples were incubated at 37° C. for 0, 24, 48, 72, 96, or 240 hours.Samples removed from each time point were transferred to −80° C. forstorage. Analysis was performed using a label-free bio-layerinterferometry assay. Matrix samples were diluted to 1:20 in 1×PBScontaining 0.05% Tween-20 and 1% BSA (assay buffer). Control samples ofMORAb-109 DAR 0, DAR 1, DAR 2, and DAR 6 were diluted to 0.1 mg/mL inmatched matrix. Negative control samples were 5% matrix-alone.Biotinylated mesothelin at 5 μg/mL in assay buffer was captured on SAstreptavidin biosensor tips (300 sec; Pall-ForteBio), followed bycapture of diluted stability samples and controls (300 sec). Payload wasthen quantitated by binding of rabbit-human chimeric anti-eribulinantibody 5E4 at 100 mg/mL. Association was monitored for 300 sec, atwhich point binding had reached equilibrium. Binding level at the end ofdissociation phase (R_(eq)) was determined for each sample at 295 sec ofassociation. Stability was determined by plotting percent R_(eq)relative to t₀, where:percent R _(eq) =R _(eq) t _(x) /R _(eq) t ₀[100] and t _(x)=0-240hours.2.9 In Vitro Cytotoxicity Analysis

A431, A3, OVCAR3, HEC-251, and H226 cells were sub-cultured and seededat 5,000 cells/well in complete growth medium in 96-well tissue cultureplates, and incubated at 37° C., 5% CO₂ overnight (16 hours). Testreagents were serially diluted 1:3 in 2 mL deep-well dilution plates,starting at 200 nM (10 dilutions total). Diluted samples (1004) wereadded to the cell plates (starting concentration of test samples at 100nM). Plates were incubated at 37° C., 5% CO₂ for an additional 5 days.Medium was then discarded, and plates were washed once with 200 μL DPBS,stained with 50 μL of 0.2% Crystal Violet solution at room temperaturefor 15 min, and then washed extensively with tap water. Plates wereair-dried, and Crystal Violet was dissolved with 200 μL of 1% SDSsolution. Plates were read at 570 nm. Data was analyzed using GraphPadPrism 6.

2.10 Results

2.10.1 Initial Screening of Humanized Anti-Mesothelin Eribulin ADCs

Fifteen anti-mesothelin antibodies were sub-cloned, scale-up expressedand purified, and conjugated using maleimide-VCP-eribulin as payload atthe Cys80 position. All ADCs were purified and characterized usingSEC-HPLC for aggregation analysis, HIC-HPLC for DAR analysis, andcell-based assay with A431-A3 (MSLN^(hi)), A431 (MSLN^(lo)), and OVCAR3(MSLN^(hi)) cell lines. The cells were treated with ADC for 6 hours thenwashed off, or treated for 48 hours (A431-A3 and A431 cells) or 72 hours(OVCAR3 cells) for the potency comparison. Characterization data issummarized in

Table 17. Based on the characterization data below, six antibodies(bolded) were selected for humanization.

TABLE 17 Characterization of fifteen anti-mesothelin eribulin ADCsSEC-HPLC- SEC-HPLC 15% IPA % % Cell based Cytotoxicity assay, EC50 (nM)Sample Conc. Monomer Monomer A3- A3- A431- A431- OVCAR3- OVCAR3- No.Name (mg/mL) Lot DAR mAb ADC 6 hr 48 hr 6 hr 48 hr 6 hr 72 hr 133O11-VCP- 0.46 02750-83G 2.00 95.4 97.58 0.90 0.50 >100 >100 2.74 4.59Eribulin 2 111B10-VCP- 0.98 02750-83H 2.00 89.9 93.69 0.300.14 >100 >100 3.11 3.15 Eribulin 3 324O5-VCP- 0.97 02750-83I 2.00 83.585.92 1.15 0.72 >100 >100 3.33 2.40 Eribulin 4 178F16-VCP- 0.9902750-83J 2.00 81.4 84.54 0.65 0.38 >100 >100 1.36 0.08 Eribulin 5237N18-VCP- 0.83 02750-83K 2.00 92.8 95.84 0.51 0.24 >100 >100 14.654.41 Eribulin 6 383I18-VCP- 0.71 02750-84I 1.83 93.5 95.80 1.060.83 >100 >100 16.25 2.16 Eribulin 7 393L14-VCP- 93.0 93.88 1.701.18 >100 >100 13.37 4.04 Eribulin 8 62B10-VCP- 0.48 02750-84J 2.00 98.746.84 0.64 0.33 >100 >100 11.70 1.84 Eribulin 9 55B4-VCP- 0.42 02750-84K2.00 98.4 73.44 0.55 0.27 >100 >100 32.17 1.33 Eribulin 10 120N18-VCP-0.3 02750-83G 2.00 97.4 61.42 1.67 0.98 >100 >100 2.16 0.20 Eribulin 11201C15-VCP- 0.61 02750-83H 2.00 94.3 95.84 0.71 0.54 >100 >100 2.83 0.05Eribulin 12 346C6-VCP- 0.9 02750-83I 2.00 91.4 92.28 0.95 0.29 >100 >1008.20 0.49 Eribulin 13 264E24-VCP- 0.66 02750-83J 1.63 46.2? 73.67 1.040.74 >100 >100 1.71 0.14 Eribulin 14 345A12-VCP- 0.4 02750-83K 2.00 95.798.53 0.95 0.80 >100 >100 0.66 0.09 Eribulin 15 102A6A-VCP- 0.1902750-83D 2.00 98.1 67.87 0.38 0.21 >100 >100 1.92 0.12 Eribulin 16102A6B-VCP- 0.31 02750-83L 2.00 97.9 19.02 0.84 0.53 >100 >100 1.85 0.14Eribulin 17 1552-VCP- 0.63 02750-83F 1.89 97.1 97.20 >10058.78 >100 >100 >100 10.60 Eribulin 18 Eribulin 6 mM 4.08 1.09 2.86 0.002.81 0.252.10.2 HC1-LC1 Humanization and In Vitro Cytotoxicity of ADCs

The sequences for the rabbit 102A6A2, 11B10, 201C15, 345A12, and 346C6Fv regions were BLASTed for the closest homology to human germlinevariable domain protein sequences using IGBLAST (National Center forBiotechnology Information (NCBI)) and IMGT/DomainGapAlign (InternationalImMunoGeneTics Information System (IMGT®)) tools. Rabbit frameworksequences were replaced with the closest homologous human germlinesequences to generate CDR-grafted humanized variants (HC1 and LC1). Thefinal two residues of Kabat-defined FWRH2 were retained as rabbitresidues. The final Kabat defined FWRH3 residue was retained for 111B10.The RESPECT-L motif Cys80 and Ala83 in the Vκ region was retained forall clones. After the humanized antibodies were generated, both chimericand humanized antibodies were conjugated with three different payloads(maleimide-VCP-eribulin, maleimide-VCP-cryptophycin, andmaleimide-VCP-eribulin dimer) varying the hydrophobicity. Bindingaffinity to mesothelin was measured by BIAcore for all antibodies, andADCs were characterized for percent (%) aggregation, DAR and in-vitropotency, as summarized in Table 18. Potency payloads of humanizedanti-mesothelin ADCs were also measured and are summarized in Table 19.ADCs had low nanomolar cell killing EC50 values in all five cell linestested.

TABLE 18 Characterization summary for lead chimeric and humanizedanti-mesothelin ADCs Parental mAb Affinity ADC Epitope k_(a) k_(d) K_(D)Payload HIC-Ethyl Bin (10⁵ M⁻¹ sec⁻¹) (10⁻³ sec⁻¹) (10⁻⁹ M) Drug-linkerDAR 33O11 xi 1 VCP-eribulin 1.92 VCP-cryptophycin 1.87 VCP-eribulindimer 1.17 zu 2.2 0.65 3.4 VCP-eribulin 1.69 VCP-cryptophycin 1.33VCP-eribulin dimer 1.63 111B10 xi 2 6.5 3.9 6.3 VCP-eribulin 1.90VCP-cryptophycin 1.86 VCP-eribulin dimer 1.85 zu 5.1 3 6.5 VCP-eribulin1.81 VCP-cryptophycin 1.76 VCP-eribulin dimer 1.78 201C15 xi 1 2.4 0.261.1 VCP-eribulin 1.85 VCP-cryptophycin 1.75 VCP-eribulin dimer 0.96 zu3.1 1.1 4.2 VCP-eribulin 1.80 VCP-cryptophycin 1.74 VCP-eribulin dimer1.75 346C6 xi 3 3.8 0.49 1.4 VCP-eribulin 1.56 VCP-cryptophycin 1.52VCP-eribulin dimer 1.60 zu 133 93 8.9 VCP-eribulin 1.63 VCP-cryptophycin1.65 VCP-eribulin dimer 1.68 345A12 xi 5 26 0.42 0.12 VCP-eribulin n/aVCP-cryptophycin n/a VCP-eribulin dimer n/a zu 35 2.1 0.2 VCP-eribulin1.72 VCP-cryptophycin 1.60 VCP-eribulin dimer 1.59 102A6A2 xi 7 n.b. n.bn.b VCP-eribulin n/a VCP-cryptophycin n/a VCP-eribulin dimer n/a zu n.b.n.b n.b VCP-eribulin 1.87 VCP-cryptophycin 1.87 VCP-eribulin dimer 1.68155D5 xi VCP-eribulin dimer 1.62 Eribulin n/a Cryptophycin n/a VCP-DiOHEribulin Dimer n/a ADC SEC-HPLC Cell-Based Cytotoxicity Assay, EC50 (nM)% aggregate % monomer A431 OVCAR3 HEC-251 H226 A3 33O11 xi 8.97 91.0340.67 0.008 3.950 >100 0.14 12.74 87.26 6.79 0.010 0.110 0.06 0.03 32.7067.30 1.40 0.030 0.33 0.93 0.05 zu 1.42 98.58 ~100 0.064 26.500 >1000.28 0.90 99.10 22.50 0.066 5.35 5.81 0.04 1.08 98.92 2.01 0.025 0.310.22 0.04 111B10 xi 4.25 95.75 38.10 0.004 13.960 ~100 0.05 8.83 91.1710.93 0.011 1.600 2.66 0.016 9.25 90.75 0.96 0.007 0.06 0.71 0.011 zu3.64 96.36 68.92 0.014 27.42 >100 0.12 1.80 98.20 4.30 0.011 0.820 1.360.015 4.47 95.53 1.68 0.007 0.13 1.15 0.025 201C15 xi 1.62 98.38 48.500.004 14.82 ~100 0.27 2.10 97.90 8.08 0.012 0.540 1.02 0.12 0.00 100.000.63 <0.003 0.10 0.64 0.065 zu 5.84 94.16 68.88 0.290 20.42 >100 0.4111.51 88.49 2.55 0.120 0.600 1.73 0.037 9.94 90.06 1.12 0.063 0.28 1.260.082 346C6 xi 5.28 94.72 34.49 0.087 5.73 ~100 0.11 8.28 91.72 4.180.042 0.190 1.21 0.043 10.43 89.57 1.32 0.026 0.09 0.99 0.035 zu 4.4895.52 72.86 1.180 32.54 >100 0.55 13.70 86.30 2.30 0.380 0.800 2.21 0.134.86 95.14 1.36 0.140 0.34 1.86 0.13 345A12 xi n/a n/a n/a n/a n/a n/an/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a zu 4.2495.76 63.49 0.004 20.70 >100 0.091 6.17 93.83 3.03 <0.003 0.350 0.690.01 5.05 94.95 1.04 <0.003 0.06 0.79 0.016 102A6A2 xi 89.50 10.50 36.180.024 2 >100 0.12 100.00 0.00 9.52 0.110 0.280 0.21 0.11 n/a n/a n/a n/an/a n/a n/a zu 3.60 96.40 57.44 0.046 8.67 >100 3.02 3.64 96.36 3.540.021 0.054 0.08 0.0092 1.74 98.26 1.40 0.047 0.05 0.50 0.018 155D5 xi14.42 81.63 1.20 0.210 0.47 1.50 1.41 n/a n/a 0.47 0.11 0.2 5.3 1.03 n/an/a 0.8 0.24 0.43 1.94 1.01 n/a n/a 0.03 <0.003 0.003 0.32 0.08

TABLE 19 In-vitro cell based potency of payloads Cell-Based CytotoxicityAssay EC50 (nM) A431 OVCAR3 HEC-251 H226 A3 Eribulin 0.47 0.11 0.2 5.31.03 Cryptophycin 0.8 0.24 0.43 1.94 1.01 VCP-DiOH Eribulin Dimer 0.03<0.003 0.003 0.32 0.082.10.3 Humanization Refinement

Due to loss of mesothelin binding for 201C15, 345A12, and 346C6 clones,subsequent mutations were required to retain binding to mesothelin. Therabbit and CDR-grafted Fv sequences were used to generate in silicomodels of the variable domains. The theoretical structure of the rabbitand humanized models were superimposed, and residues in close proximityto the CDRs were analyzed for potential structural influence on theoverall structure of the CDR loops. The residues differing between therabbit and humanized sequences were identified. Most of the differingresidues were not located at the dimer interface or were distal to theCDRs. Several residues in the VH and Vκ regions were found to be inclose proximity (within 5 Å) of the CDRs, and were further analyzed.

Two humanized regions in the VH region were identified as possiblyinterfering with antigen-binding in clones 201C15, 345A12, and 346C6.The N terminus for all clones was one amino acid longer in HC1 than inthe rabbit sequence. Also, each had a 2-amino acid deletion in FWRH3(residues 72-73). For each of these clones, the first five amino acidsand the six amino acids surrounding the FWRH3 deletion (residues 71-76)of HC1 were reverted to the rabbit sequences. Residue 93 of 345A12 wasalso reverted to rabbit in HCS. Regarding LC1, the N termini of 201C15,345A12, and 346C6 were reverted to the rabbit sequence. One residue inFWRL3 of 20105 (residue 67) and 345A12 (residue 70) was identified aspotentially interacting with the CDRs and one residue in FWRL2 of 346C6(residue 36) was likewise identified.

2.10.4 Super-Humanizing 345A12

With the identification of additional rabbit residues in Vκ as criticalfor antigen-binding, further mutants of 345A12 were generated tointroduce increasing numbers of human residues throughout the VH and Vκregions. Analysis of the in silico model identified residues 35, 48, 49,57, 58, 61, 62, 63, and 64 in VH and residues 1, 3, 24, 55, and 70 inVK.

2.10.5 Biophysical Characterization of Super-Humanized 345A12 Antibodies

The super-humanized 345A12 antibodies were purified from 350 mL ofscale-up cell culture and formulated in 1×DPBS. The antibodies wereconcentrated for physical-chemical property evaluation. As shown inTable 20, the combination of HC10-LC7 and HC15-LC7 were precipitatedduring concentration step, potentially due to relative low pI or poorsolubility. Purified antibodies were analyzed by BIAcore for mesothelinbinding affinity, and the data are summarized in Table 21 below.

TABLE 20 Summary of purified humanized 345A12 antibodies mAb-VCP-DiOHSupernatant Purification Eribulin Dimer, mAb-VCP- Concentration to mAbsVolume (mL) Yield yield Eribulin, Yield 5 mg/mL 345A12- 350 mL 77.5 mg @3.9 mg @ 1.3 11 mg @ 5.0 Yes HC10-LC4 5.1 mg/mL mg/mL, 78% mg/mL, 73%345A12- 350 mL 78 mg @ 4.5 mg @ 1.6 8.1 mg at 2.7 No, precipitatedHC10-LC7 5.2 mg/mL mg/mL, 90% mg/mL, 54% 345A12- 350 mL 191 mg @ 4.2 mg@ 1.4 11 mg @4.85 Yes HC15-LC4 8.3 mg/mL mg/mL, 84% mg/mL, 73% 345A12-350 mL 87.2 mg @ 3.3 mg @1.1 4.6 mg @ 1.54 No, precipitated HC15-LC7 4.0mg/mL mg/mL, 66% mg/mL, 31%

TABLE 21 Binding affinity to mesothelin for humanized 345A12 AffinityAggregation Conjugation Run 1 Run 2 % Monomer % Conjugation k_(a) k_(d)K_(D) k_(a) k_(d) Chimeric 2.63E+06 4.64E−04 1.76E−10 HC1-LC2 88.85 1.62.59E+06 4.32E−04 1.67E−10 2.47E+06 3.04E−04 HC1-LC4 82.96 1.33 2.35E+062.57E−04 HC1-LC7 87.23 1.29 HC10-LC4 80.73 1.32 HC10-LC7 82.75 1.21HC15-LC4 86.56 1.3 HC15-LC7 92.7 1.42 Affinity Run 2 Run 3 Run 4 K_(D)k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) Chimeric HC1-LC2 1.13E−10 HC1-LC41.18E−10 1.96E+06 1.30E−04 6.61E−11 1.35E+06 1.61E−04 1.19E−10 HC1-LC71.83E+06 1.40E−04 7.66E−11 1.38E+06 1.79E−04 1.29E−10 HC10-LC4 1.80E+061.52E−04 8.47E−11 1.21E+06 1.80E−04 1.49E−10 HC10-LC7 1.26E+06 1.89E−041.50E−10 HC15-LC4 1.44E+06 1.12E−04 7.79E−11 HC15-LC7 1.49E+06 1.16E−047.82E−112.10.6 DSC and cIEF Analyses

The thermal melting curves of F(ab′)2 fragments were analyzed by DSC.The profiles of the HC15-LC4 F(ab′)2 and HC10-LC4 F(ab′)2 are shown inFIG. 8 .

The pI of 345A12-HC10-LC4 and 345A12-HC15-LC4 mAbs were analyzed bycIEF. The pI varied within 0.06 pH units between each mAb, 8.19 for345A12-HC10-LC4 and 8.25 for 345A12-HC15-LC4 (Table 22).

TABLE 22 cIEF analysis % Acidic % Neutral % Basic Sample Name pI PeaksPeak Peaks 345A12-HC10-LC4 8.19 24.412 57.171 18.417 345A12-HC15-LC48.25 34.787 50.790 14.4242.10.7 Serum Stability

PBS/human serum stability evaluation of 345A12-HC10-LC4 and345A12-HC15-LC4 ADCs were tested for up to 10 days, as described inSection 2.7. Data are summarized in Table 23 below.

TABLE 23 In vitro PBS/human serum stability of zu345A12-VCP-eribulinADCs, HC15-LC4 vs. HC10-LC4 Total Ab Intact ADC Adjusted % AdjustedSample Mean Conc. Result Difference Mean Conc. Result % Difference #Sample Description Dilution (ng/mL) % CV (μg/mL) from T = 0 (ng/mL) % CV(μg/mL) from T = 0 1 Zu345A12-HC10LC4- 1:2,000 231 2.14 462 N/A 258 1.42516 N/A VCP-Eribulin T = 0, in human serum 2 Zu345A12-HC10LC4- 1:2,000221 4.59 442 −4.3 190 1.70 380 −26.4 VCP-Eribulin T = 24, in human serum3 Zu345A12-HC10LC4- 1:2,000 208 0.840 416 −10.0 154 3.19 308 −40.3VCP-Eribulin T = 48, in human serum 4 Zu345A12-HC10LC4- 1:2,000 213 1.46426 −7.8 142 1.66 284 −45.0 VCP-Eribulin T = 72, in human serum 5Zu345A12-HC10LC4- 1:2,000 210 2.55 420 −9.1 105 21.5 210 −59.3VCP-Eribulin T = 96, in human serum 6 Zu345A12-HC10LC4- 1:2,000 207 4.27414 −10.4 97.0 6.31 194 −62.4 VCP-Eribulin T = 240, in human serum 7Zu345A12-HC15LC4- 1:2,000 284 0.387 568 N/A 278 4.84 556 N/AVCP-Eribulin T = 0, in human serum 8 Zu345A12-HC15LC4- 1:2,000 282 4.19564 −0.7 229 2.71 458 −17.6 VCP-Eribulin T = 24, in human serum 9Zu345A12-HC15LC4- 1:2,000 273 0.103 546 −3.9 180 1.87 360 −35.3VCP-Eribulin T = 48, in human serum 10 Zu345A12-HC15LC4- 1:2,000 2580.744 516 −9.2 144 0.436 288 −48.2 VCP-Eribulin T = 72, in human serum11 Zu345A12-HC15LC4- 1:2,000 270 0.669 540 −4.9 135 3.13 270 −51.4VCP-Eribulin T = 96, in human serum 12 Zu345A12-HC15LC4- 1:2,000 2701.48 540 −4.9 111 0.669 222 −60.1 VCP-Eribulin T = 240, in human serum13 Zu345A12-HC10LC4- 1:2,000 180 1.03 360 N/A 229 1.40 458 N/AVCP-Eribulin T = 0, in PBS 14 Zu345A12-HC10LC4- 1:2,000 184 0.583 3682.2 187 2.07 374 −18.3 VCP-Eribulin T = 24, in PBS 15 Zu345A12-HC10LC4-1:2,000 166 3.12 332 −7.8 177 1.64 354 −22.7 VCP-Eribulin T = 48, in PBS16 Zu345A12-HC10LC4- 1:2,000 180 0.829 360 0.0 177 2.00 354 −22.7VCP-Eribulin T = 72,in PBS 17 Zu345A12-HC10LC4- 1:2,000 179 1.73 358−0.6 174 3.87 348 −24.0 VCP-Eribulin T = 96, in PBS 18 Zu345A12-HC10LC4-1:2,000 171 0.915 342 −5.0 131 6.24 262 −42.8 VCP-Eribulin T = 240, inPBS 19 Zu345A12-HC15LC4- 1:2,000 225 1.14 450 N/A 233 0.325 466 N/AVCP-Eribulin T = 0, in PBS 20 Zu345A12-HC15LC4- 1:2,000 226 1.45 452 0.4209 3.53 418 −10.3 VCP-Eribulin T = 24, in PBS 21 Zu345A12-HC15LC4-1:2,000 233 0.754 466 3.6 210 0.942 420 −9.9 VCP-Eribulin T = 48, in PBS22 Zu345A12-HC15LC4- 1:2,000 210 4.03 420 −6.7 186 2.11 372 −20.2VCP-Eribulin T = 72, in PBS 23 Zu345A12-HC15LC4- 1:2,000 247 10.4 4949.8 182 0.0585 364 −21.9 VCP-Eribulin T = 96, in PBS 24Zu345A12-HC15LC4- 1:2,000 226 0.514 452 0.4 145 6.91 290 −37.8VCP-Eribulin T = 240, in PBS2.10.8 Matrix Stability of 345A12-HC15-LC4-VCP-Eribulin in VariousMatrices Using a DAR-Sensitive Octet Assay

345A12-HC15-LC4-VCP-eribulin (DAR2) was analyzed for in vitro stabilityin mouse, rat, cynomolgus monkey, and human plasma and serum. ADC wasincubated in matrix at 0.1 mg/mL for 1 week, and time points wereremoved after 1, 2, 3, 4, and 10 days. Analysis was done using aDAR-sensitive Octet (biolayer inferometry)-based assay as described inSection 2.3.1. Results are shown in FIG. 9 .345A12-HC15-LC4-VCP-eribulin (DAR2) demonstrated a time-dependentrelease of payload, with an average of 20% release after a 10 dayincubation at 37° C.

2.10.9 Cultures Producing Rabbit IgG and Polyclonal Antibodies AgainstHuman Mesothelin

On week 2, the wells producing rabbit IgG antibody were identified byIgG FRET using europium cryptate. Wells producing IgG were screened forthe presence of rabbit IgG Fcγ antibody by ELISA against plates coatedwith 1 μg/mL of CHO-MT40 mesothelin. The cultures producing mesothelinspecific rabbit IgG were confirmed by ELISA screening against 1 μg/mL ofmesothelin and counter screened against 1 μg/mL of CD73-his. FRET andELISA were performed on the Biomek® FX robotic system (Beckman).

Example 3: In Vivo Studies

ADCs comprising lead humanized anti-mesothelin antibodies and eribulinconjugates were evaluated in mice using human lung and gastric cancerxenograft models and a human mesothelioma patient-derived xenograft(PDX) model according to the protocol described below. The anti-canceractivity and off-target toxicity of different DAR species of the ADCswas assessed.

3.1 Reagents and Materials

3.1.1 Antibodies

The antibodies used in the following studies have an unpaired cysteineat light chain position 80 (LCcys80) and include both the rabbit-humanchimeric (−xi) and humanized (−zu) forms of the anti-human mesothelinantibodies xi345A12-HC1-LC2, xi102A6A2-HC1-LC2, zu345A12-HC1-LC2,zu345A12-HC10-LC4, and zu345A12-HC15-LC4.

3.1.2 Conjugatable Cytotoxins and LCcys80 ADCs

Linker-cytotoxin compounds used in the following studies includemaleimide-VCP-eribulin and maleimide-VCP-diOH eribulin dimer.

3.1.3 Tumor Cell Lines

Human NSCLC cell line NCI-H2110, human gastric cancer cell line NCI-N87,and human mesothelioma cancer cell line HAY were used in the followingstudies. All cell lines used were obtained directly from the AmericanType Culture Collection (ATCC), with the exception of HAY cells, whichwere obtained from NCI.

3.1.4 Other Reagents

All reagents used were obtained from commercial suppliers atresearch-grade or higher, unless otherwise indicated.

3.2 In Vivo Screening and Efficacy Studies in Human Cancer XenograftModels

3.2.1 Study Animals

Female CD-1 IGS mice (Charles River, 7-9 weeks old) were used for themaximum tolerated dose (MTD) study, female NOD.CB17-SCID mice (JacksonLaboratory) were used for the NCI-H2110 and HAY xenograft studies, andfemale NCr nude mice (Taconic, 5 weeks old) were used for the NCI-N87xenograft studies. Upon arrival, animals were acclimated for 5-7 daysprior to inoculation. Animals were housed 3-5 mice per ventilated cagewith sterilized food pellets and water bottle available ad lib. Animalswere ear tagged and weighed prior to study initiation.

3.2.2 Cell Culture

Cryopreserved NCI-H2110, NCI-N87, or HAY cells from frozen stocks werecultured in medium containing necessary supplements. Cells weresub-cultured in complete medium for 2 passages before being used for invivo inoculation.

3.2.3 Tumor Implantation, Enrollment Process, and Treatment

Cells were suspended in PBS mixed with ice-cold Matrigel at 1:1(vol:vol) to a final concentration of 1.0×10⁸ cells/mL or 5.0×10⁷cells/mL for the NCI-H2110 and HAY cells. Mice were injectedsubcutaneously with 100 μL/mouse of cell mixture and monitored for bodyweight and tumor growth. Measurements were taken by digital caliper 3times weekly beginning on day 3 post-implantation.

3.2.4 Tumor Measurement and Treatment

Tumor volume was calculated using the formula: W (mm)×L (mm)×D (mm)×n/6.Mice were randomized to five mice per group once tumor implants reachedan average volume of 100 mm³. Treatment was given intravenously at avolume of 200 μL. Terminal body weight was measured and recorded at theend of each study.

3.2.5 Statistical Analysis

Tumor volumes of animals from each treatment group were compared with acontrol group by a repeated-measures two-way ANOVA, followed by aBonferroni post-test. Comparison of tumor growth within eachexperimental group was also performed using the same statisticalanalysis.

3.3 In Vivo Efficacy Studies in a Human Mesothelioma PDX Model

3.3.1 Study Animals

NMRI nu/nu female mice (Janvier Labs, 5-6 weeks) were acclimated for atleast 4 days upon arrival prior to inoculation. Animals was housed 3-5mice per ventilated cage with sterilized food pellets and water bottleavailable ad lib. Animals were ear marked and weighed prior to studyinitiation.

3.3.2 Xenotransplantation

On day 0 of the study, Meso 7212 tumors were removed from five donormice under sterile conditions. Donor tumor tissue was cut into 2×2 mmfragments and placed in a sterile Petri dish covered with 0.9% saline.In parallel, the receptor animals were subcutaneously treated with theanalgesic Metacam® (2 mg/kg) and then anaesthetized by a singleintravenous injection (0.15 mL/mouse) with Etomidat-Lipuro® (12 mg/kg).A superficial vertical incision in the skin of 5-8 mm on the left flankwas performed. The tip of a surgical scissor was inserted into theincision, directly over the flank, and was used to form a pocket in thesubcutaneous space. One tumor fragment per mouse was implanted into thepocket using surgical tweezers. The incision was closed with a metalclip and the animals were placed back into a clean cage.

3.3.3 Experimental Procedure

During tumor propagation, tumor diameters were measured using a digitalcaliper (Mitutoyo). Animals were randomly assigned into experimentalgroups according to their tumor volume (inclusion criteria for tumorvolume, 0.1-0.3 cm³). Tumor volumes and body weights were recorded twiceweekly.

3.3.4 Treatment

Eribulin was administered intravenously at doses of 0.2, 0.3, and 3.2mg/kg on the day of randomization. MORAb-109 (DAR 0, 2, and 6) wasadministered intravenously at a dose of 10.0 mg/kg on the day ofrandomization or a dose of 2.5 mg/kg on four consecutive days. Theadministration volume was 10 mL/kg for intravenous injections throughoutall experimental groups.

3.3.5 Statistical Analyses

Descriptive statistics were performed on the data for tumor volume andbody weight. Tumor volumes of animals from each treatment group werecompared with the control group by using a repeated-measures two-wayANOVA, followed by a Bonferroni post-test. Additionally, the comparisonof tumor growth of animals within each group was also performed with thesame statistical analysis.

3.4 Results—In Vivo Screening and Efficacy Studies

3.4.1 Study M109-004-2016: In Vivo Screening of 345A12-HC1-LC2 and102A6A2 HC1-LC2 Eribulin Dimer ADCs in a Human NSCLC Xenograft Model

Comparative in vivo screening of two clones of anti-mesothelinantibodies (345A12-HC1-LC2 and 102A6A2-HC1-LC2) was performed in a humannon-small cell lung cancer (NSCLC) NCI-H2110 xenograft model. Mice weretreated with 345A12-HC1-LC2-diOH eribulin dimer ADC at 2.5 mg/kg or102A6A2-HC1-LC2-diOH eribulin dimer ADC at 2.5 mg/kg. Anti-tumoractivity and body weight changes for both ADCs are shown in FIG. 10A andFIG. 10B, respectively.

3.4.2 Study M109-006-2017: Preliminary Evaluation of Maximum ToleranceDose (MTD) of 345A12-HC1-LC2 and 345A12-HC15-LC4 Eribulin Dimer ADCs inCD-1 Mice

Body weight changes of female CD-1 mice were measured followingadministration of 5, 10, 15, or 20 mg/kg of 345A12-HC1-LC2-diOH eribulindimer ADC, or 5, 10, or 20 mg/kg of 345A12-HC15-LC4-diOH eribulin dimerADC. Body weight changes for each ADC are shown in FIG. 11A and FIG.11B, respectively.

3.4.3 Study M109-007-2017: In Vivo Screening of 345A12-HC10-LC4 and345A12-HC15-LC4 Eribulin Dimer ADCs in a Human NSCLC Xenograft Model

Comparative in vivo screening of two additional clones ofanti-mesothelin antibodies (345A12-HC10-LC4 and 345A12-HC15-LC4) wasperformed in a human NSCLC NCI-H2110 xenograft model. Mice were treatedwith 345A12-HC10-LC4-diOH eribulin dimer ADC at 2.5 mg/kg or345A12-HC15-LC4-diOH eribulin dimer ADC at 2.5 mg/kg. Anti-tumoractivity and body weight changes for both ADCs are shown in FIG. 12A andFIG. 12B, respectively.

The 345A12-HC15-LC4 clone was selected based on its anti-tumor activityand toxicity profile as the candidate clone for the antibody used in theMORAb-109 ADC.

3.4.4 Study M109-010-2018: Anti-Tumor Effect of DAR2 and DAR6 Species ofthe 345A12-HC15-LC4-VCP-Eribulin ADC (MORAb-109) in a Human GastricCancer Xenograft Model

Two DAR species (DAR2 and DAR6) of the 345A12-HC15-LC4-VCP-eribulin ADC(MORAb-109) were compared in a human gastric cancer NCI-N87 xenograftmodel at a dose of 10 mg/kg. Both DAR2 and DAR6 ADC species demonstrateddurable and similar anti-tumor responses (FIG. 13A), with little to noweight loss observed following administration of either DAR species(FIG. 13B).

3.4.5 Study M109-010-2018: Anti-Tumor Effect of DAR2 and DAR6 Species ofthe 345A12-HC15-LC4-VCP-Eribulin ADC (MORAb-109) in a Human MesotheliomaXenograft Model

Two DAR species (DAR2 and DAR6) of the 345A12-HC15-LC4-VCP-eribulin ADC(MORAb-109) were compared in a human mesothelioma HAY xenograft model.Both DAR2 and DAR6 ADC species demonstrated durable and similaranti-tumor responses in mice treated with a single dose (5 mg/kg) ofMORAb-109, while eribulin alone (administered at the MTD (3.2 mg/kg) orat an equivalent molar amount of eribulin as found in MORAb-109 (0.1mg/kg)) showed limited anti-tumor effects (FIG. 14A). Acute andtemporary body weight loss was observed in mice treated with the MTDdose of eribulin, while no body weight loss was observed in mice treatedwith either ADC (FIG. 14B).

3.4.6 Anti-Tumor Effect of MORAb-109 (DAR6) in a Human Mesothelioma PDXModel

The anti-tumor effect of the 345A12-HC15-LC4-VCP-eribulin ADC(MORAb-109) (DAR6) were investigated in a human mesothelioma PDX model,Meso7212 (MV15369). Two different treatment regimens were tested: asingle administration of 10 mg/kg MORAb-109 or four consecutive dailyadministrations of 2.5 mg/kg. Both treatment regimens of MORAb-109demonstrated durable and comparable anti-tumor responses, while theequivalent molar amount of eribulin alone (0.2 mg/kg) showed limitedanti-tumor effects (FIG. 15A). Both MORAb-109 ADC treatments showedsignificantly increased anti-tumor activity as compared to the MTD doseof eribulin alone (P<0.05). Body weight changes for all treatments areshown in FIG. 15B.

3.4.7 Anti-Tumor Effect of MORAb-109 (DAR2 and DAR6) in a HumanMesothelioma PDX Model

The anti-tumor effect of two DAR species (DAR2 and DAR6) of the345A12-HC15-LC4-VCP-eribulin ADC (MORAb-109) were investigated in ahuman mesothelioma PDX model, Meso7212 (MV16071), at a singleadministration of 10 mg/kg. Both DAR2 and DAR6 species of MORAb-109demonstrated durable and comparable anti-tumor responses, while theequivalent molar amount of eribulin alone (0.3 mg/kg) and theno-eribulin conjugated MORAb-109 species (DAR0) showed limited or noanti-tumor effects (FIG. 16A). No body weight loss observed in any group(FIG. 16B). There was no statistical difference of anti-tumor effectbetween the DAR2 and DAR6 species of MORAb-109.

Example 4: Mesothelin (MSLN) Expression and In Vitro Potency

4.1 Methods

Cytotoxicity: Cells were sub-cultured and seeded at 5,000 cells/well incomplete growth medium in 96-well tissue culture plates, and incubatedat 37° C., 5% CO₂ overnight (16 hours). Test reagents were seriallydiluted 1:3 in 2 mL deep-well dilution plates, starting at 200 nM (10dilutions total). Diluted samples (100 μL) were added to the cell plates(starting concentration of test samples at 100 nM). Plates wereincubated at 37° C., 5% CO₂ for an additional 5 days. Medium was thendiscarded, and plates were washed once with 200 μL DPBS, stained with 50μL of 0.2% Crystal Violet solution at room temperature for 15 min, andthen washed extensively with tap water. Plates were air-dried, andCrystal Violet was dissolved with 200 μL of 1% SDS solution. Plates wereread at 570 nm. Data was analyzed for IC₅₀ determination using GraphPadPrism 6. Correlation analysis was performed in GraphPad Prism using anon-parametric Spearman analysis.

4.2 Results

A correlation between potency of MORAb-109 (DAR6) and mesothelinexpression was observed for all cell lines (FIG. 17 ; Tables 24 and 25).

For MORAb-109 (DAR2), when cell lines having lower mesothelin expression(FACS staining of mean fluorescence intensity (MFI)<80) were excludedfrom the analysis, a significant correlation between potency andmesothelin expression was observed (FIG. 17 ; Tables 24 and 25). Potencyof MORAb-109 (DAR2) correlated with mesothelin expression at highermesothelin expression levels.

TABLE 24 Mesothelin expression and potency correlation analysis (DAR2and DAR6) MSLN (MFI) MSLN (MFI) MSLN (MFI) MSLN (MFI) vs. vs. vs vsEribulin MORAb109 MORAb109(DAR6) MORAb109_MSLN high Spearman r r −0 1113−0 2906 −0 6773 −0 6267 95% confidence interval −0.3935 to 0.1902−0.5472 to 0 01591 −0.8093 to −0.4803 −0 8502 to −0.2118 P value P(two-tailed) 0.4566 0.0557 <0.0001 0.0054 P value summary ns ns **** **Exact or approximate P value? Approximate Approximate ApproximateApproximate Significant? (alpha = 0.05) No No Yes Yes Number of XY Pairs47 44 48 18

TABLE 25 Cell lines used in mesothelin expression and potencycorrelation analysis In-vitro potency, IC50, nM MSLN MORAb109 MORAb109Cell lines (MFI) Eribulin (DAR2) (DAR6) EGI-1 14.8 1.48 187.8 15.45 TFK1132.81 1.27 5.65 SNU-245 23.9 78.24 355.8 SNU-478 53.8 1.58 227.5 55.16SNU-1196 40.2 1.14 128.4 6.49 T-47D 4.4 0.68 95.86 22.04 JIMT-1 36.40.39 61.8 11.4 HCC1806 189.3 0.37 35.54 0.24 HEC-1-A 16.5 0.68 140.723.7 HEC-1-B 16.9 0.54 66.29 1.6 HEC-251 53.3 0.5 67.15 0.67 MFE-28045.1 3.24 196.5 164.9 NCI-H292 108.6 0.58 147.3 6.37 NCI-H322 83.9 0.4662.84 3.62 NCI-H1355 11.5 0.67 79.77 7.09 NCI-H1355-Sorted 32.7 0.796.92 6.48 NCI-H1573 17.4 60.41 6.46 NCI-H1568/MSLN 4474 0.44 1.69 1.08NCI-H1650 31.5 0.61 92.2 4.09 NCI-H1650/MSLN 1865.5 0.84 1 0.57NCI-H2110 112.2 0.32 52.23 4.17 NCI-H2126 73.6 0.53 87.89 10.82 PC911.24 0.56 85.14 15.22 H226 725.3 0.66 7.46 0.13 NCI-H23 4.42 0.6 66.7417.31 Lu65 13.13 0.56 15.85 NCI-H460 8.56 35.29 MOR/CPR 120.79 3.69 5.68A549 14.2 2.72 276 41.33 CNE2 147.7 0.49 69.55 1.64 HK1 39.2 0.89 197.55.88 HNE-1 110.8 0.87 239.2 2.03 HNE-1-T1 79.3 1.03 347 11.28 HONE1108.6 0.89 197.5 5.88 SUNE1 212.4 0.56 77.8 0.27 CaOV-3 137.6 0.44 20.930.51 COV362 113.5 2.78 193 45.29 HAC-2 172.3 26.65 228 320 Kuramochi211.2 0.89 897.8 0.1 OVCAR3 A1 220 0.15 0.005 0.005 AsPC1 33.47 1.623.13 BxPC3 15.59 0.66 86.2 5.88 Capan-2 36 1.21 138 1.99 A431 5.6 0.5571.51 14.17 A431-K5 1430.3 1.5 0.45 0.35 NCI-N87 125.8 0.11 14.7 0.1MKN1 22.06 0.96 20.14 2.89 MKN74 7.73 0.78 66.96 9.62 SNU216 94.91 1.7873.42 0.29 MKN45 57.94 3.65 100 10.39 MKN7 18.48 0.58 86.53 10.39

Example 5: Dose-Response of MORAb-109 in Human Gastric Cancer (NCI-N87)Xenograft Model

5.1 Methods

5.1.1 In Vivo Efficacy

Animals: Female NCr nude mice (Taconic), 5 weeks old at the time ofarrival were acclimated for 5-7 days prior to inoculation. Animals werehoused 3-5 mice per ventilated cage with sterilized food pellets andwater bottle available ad lib. Animals were ear tagged and weighed priorto study initiation.

Cell culture: Cryopreserved NCI-N87 cells were thawed and grown inmedium containing necessary supplements. Cells were sub-cultured incomplete medium for 2 passages before being used for in vivoinoculation.

Tumor implantation, enrollment process, and treatment: The cellsuspension in PBS was mixed with ice-cold Matrigel at 1:1 (vol:vol) to afinal concentration of 1.0×10⁸ cells/mL. 100 μL/mouse of the mixture wasinjected subcutaneously. The mice were monitored for clinical well-beingwith body weights and tumors measurements by digital caliper, 3 timesweekly, beginning on day 3 post-implantation.

Tumor measurement and treatment: Tumor volume (TV) (mm³) was calculatedusing the formula: W (mm)×L (mm)×D (mm)×n/6. When the tumors reachedaround 100 mm³ in an average, the animals were randomized to 5 pergroup. Treatment was given intravenously in a volume of 200 μL of testarticle. At the end of the study, the terminal body weight was measuredand recorded.

Statistical analysis: Descriptive statistics were performed on the dataof tumor volume and body weight. Tumor volumes of animals from eachtreatment group were compared with the control group by using therepeated-measures two-way ANOVA followed by the Bonferroni post-test.Additionally, the comparison of tumor growth of animals within eachgroup was performed with the same statistical analysis.

5.1.2 Pharmacokinetics (PK)

PK analysis was performed using both an intact ADC assay and a totalantibody assay. Total antibody refers to the sum total of all species,including conjugated and unconjugated species (i.e., DAR0+DAR1+DAR2+ . .. +DARn), whereas intact ADC refers to all conjugated species (i.e.,DAR1+DAR2+ . . . +DARn). Total antibody assay used biotinylatedmesothelin for capture. Intact ADC assay used biotinylated anti-eribulin5E4 Fab fragment for capture and AlexaFluor647-labelled anti-human Fcfor detection. Sample analysis was performed on a Gyros analyzer. Dataanalysis was performed in WatsonLIMS 7.4.1 and plotted in MicrosoftExcel.

5.2 Results

Dose-dependent efficacy was observed for dose ranges of MORAb-109 (DAR2)from 5 mg/kg to 25 mg/kg (FIGS. 18A and 18B). No body weight loss wasobserved in any dose group (FIG. 18C). A dose-dependent exposure of ADCwas observed in treated animals, as indicated by dose-dependentincreases in AUC (FIG. 19 and Table 26).

TABLE 26 PK of MORAb-109 (dose-titration) in NCI-N87 tumor-bearing miceIntact ADC Dose (mg/kg or Total Ab T_(1/2) (hr) V_(dss) (mL/kg) CL(mL/kg/hr) AUC (μg*hr/kg) C_(max) (μg/mL) 5 Intact 125.1 78.7 0.509 951262.9 Total 173.9 75.1 0.316 14375 73.2 10 Intact 148.2 99.5 0.475 19559105 Total 231.6 89.6 0.259 31631 123 15 Intact 142.1 81.1 0.414 34557169 Total 209.2 68.8 0.236 56134 205 20 Intact 151.5 90.9 0.429 43852214 Total 218.6 70.6 0.230 75718 270 25 Intact 143.5 91.1 0.463 51253271 Total 226.1 78.2 0.240 88099 322

Example 6: In Vivo Anti-Tumor Efficacy of MORAb-109 in Human OvarianCancer (OVCAR-3-A1-T1) Xenograft Model

6.1 Methods

Animals: Female NOD.CB17-SCID mice (Jackson Laboratory), 5 weeks old atthe time of arrival were acclimated for 5-7 days prior to inoculation.Animals were housed 3-5 mice per ventilated cage with sterilized foodpellets and water bottle available ad lib. Animals were ear tagged andweighed prior to study initiation.

Cell culture: Cryopreserved OVCAR-3-A1-T1 cells were thawed and grown inmedium containing necessary supplements. Cells were sub-cultured incomplete medium for 2 passages before being used for in vivoinoculation.

Tumor implantation, enrollment process, and treatment: The cellsuspension in PBS was mixed with ice-cold Matrigel at 1:1 (vol:vol) to afinal concentration of 5.0×10⁷ cells/mL. 100 μL/mouse of the mixture wasinjected subcutaneously. The mice were monitored for clinical well-beingwith body weights and tumors measurements by digital caliper, 3 timesweekly, beginning on day 3 post-implantation.

Tumor measurement and treatment: Tumor volume (TV) (mm³) was calculatedusing the formula: W (mm)×L (mm)×D (mm)×n/6. When the tumors reachedaround 100 mm³ in an average, the animals were randomized to 5 pergroup. Treatment was given intravenously in a volume of 200 μL of testarticle. At the end of the study, the terminal body weight was measuredand recorded.

Statistical analysis: Descriptive statistics were performed on the dataof tumor volume and body weight. Tumor volumes of animals from eachtreatment group were compared with the control group by using therepeated-measures two-way ANOVA followed by the Bonferroni post-test.Additionally, the comparison of tumor growth of animals within eachgroup was performed with the same statistical analysis.

6.2 Results

MORAb-109 (DAR2) demonstrated tumor growth delay in a human ovariancancer OVCAR-3-A1-T1 xenograft model (FIG. 20A and FIG. 20B).

Example 7: In Vivo Anti-Tumor Efficacy of MORAb-109 in Human NSCLC PDXModel (LC-F-25)

7.1 Methods

Animals: Outbred athymic (nu/nu) female mice (HSD:Athymic Nude-Foxn1nu),5 weeks old at the time of arrival were acclimated for at least 4 daysprior to inoculation. Animals were housed 3-5 mice per ventilated cagewith sterilized food pellets and water bottle available ad lib. Animalswere ear marked and weighed prior to study initiation.

Xenotransplantation: LC-F-25 was established as a growing tumor(P9.1.1/0) from a primary non-small cell lung adenocarcinoma from ahuman patient. LC-F-25 has lower MSLN expression in terms of percentpositivity and overall intensity, based on immunohistochemistry (IHC)analysis, relative to other tumor types (such as, e.g., LXFA-737(Example 8)).

Experimental procedure: Thirty one (31) mice with an established growingLC-F-25 tumor (P9.1.1/0) between 108 and 288 mm³ were allocated totreatment when the mean and median tumor volume reached 153.5 and 126mm³, respectively.

Treatment: Efficacy was evaluated in 4 groups of 7 to 8 mice each:

-   -   In group 1, vehicle was dosed at 5 mL/kg, i.v., single dose on        day 1.    -   In groups 2 and 3, eribulin was dosed respectively at 0.1 mg/kg        (5 mL/kg) and 3.2 mg/kg (6.4 mL/kg), i.v., single dose on day 1.    -   In group 4, MORAb-109 was dosed at 10 mg/kg (5 mL/kg), i.v.,        single dose on day 1.

Tumors were measured and mice were weighed twice a week during theexperimental period.

Statistical analysis: Statistical analysis was performed for eachmeasurement by a Mann-Whitney non-parametric comparison test. Eachtreated group was compared with the control group.

7.2 Results

MORAb-109 (DAR2) given at a single dose of 10 mg/kg by intravenous routewas well tolerated without bodyweight loss by LC-F-25 tumor-bearing mice(FIG. 21B). MORAb-109 (DAR2) demonstrated tumor growth delay at 10 mg/kgin the LC-F-25 NSCLC PDX model (FIG. 21A).

Eribulin given at a single dose of 0.1 mg/kg (the equivalent molaramount of payload in MORAb-109 when administered at 10 mg/kg) byintravenous route was well tolerated by LC-F-25 tumor-bearing mice butdid not induce statistically significant tumor growth inhibition (FIG.21A and FIG. 21B).

Eribulin given at a single dose of 3.2 mg/kg (mouse MTD dosage or 32times higher than the molar amount of eribulin in MORAb-109 whenadministered at 10 mg/kg) by intravenous route was well tolerated byLC-F-25 tumor-bearing mice, but induced slight and transient bodyweightloss from 3 to 10 days after administration (FIG. 21B). At this dose,eribulin induced statistically significant tumor growth inhibition, withpartial tumor regressions in 6 out of 8 mice (FIG. 21A).

Example 8: In Vivo Anti-Tumor Efficacy of MORAb-109 in Human NSCLC PDXModel (LXFA-737)

8.1 Methods

Animals: Female NMRI nu/nu mice (Crl:NMRI-Foxn1nu), 4 to 6 weeks of age.

Xenotransplantation: LXFA-737 was established as a growing tumor (P14N4)from a primary non-small cell lung adenocarcinoma from a human patient.LXFA-737 has moderate MSLN expression in terms of overall intensity andhigher percent positivity, based on IHC analysis, relative to othertumor types (such as, e.g., LC-F-25 (Example 7)).

Experimental procedure: Animals were monitored until the tumor implantsreached the study volume criteria of 50-250 mm³ (e.g., 80-200 mm³) in asufficient number of animals. Mice were assigned to groups, aiming atcomparable group median and mean tumor volumes. The day of randomizationwas designated as day 0.

Treatment: Efficacy was evaluated in 4 groups of 6 to 7 mice each:

-   -   In group 1, vehicle was dosed at 5 mL/kg, i.v., single dose on        day 1.    -   In groups 2 and 3, eribulin was dosed respectively at 0.2 mg/kg        and 3.2 mg/kg, i.v., single dose on day 1.    -   In group 4, MORAb-109 was dosed at 10 mg/kg, i.v., single dose        on day 1.

Tumors were measured and mice were weighed twice a week during theexperimental period. The first day of dosing was day 1, one day afterrandomization (day 0).

Statistical analysis: Inhibition of Tumor Growth, Test/Control Value in% (Min. T/C): The test versus control value (T/C in %) was calculatedfrom the ratio of the median residual tumor volume (RTV) values of testversus control groups on day X multiplied by 100. Tumor volume doublingand quadrupling time (Td, Tq) for test and control groups was defined asthe time interval (in days) required for a group to reach a median RTVof 200% or 400%.

8.2 Results

MORAb-109 (DAR2) demonstrated robust anti-tumor efficacy (minimum T/C,2.3% on day 41) at 10 mg/kg in the LXFA-737 NSCLC PDX model (FIG. 22A)and its Tq was not reached during the study. MORAb-109 given at thesingle dose was also well tolerated without bodyweight loss by LXFA-737tumor-bearing mice (FIG. 22B).

Eribulin given at a single dose of 3.2 mg/kg (mouse MTD dosage or 32times higher than the molar amount of eribulin in MORAb-109 whenadministered at 10 mg/kg) by intravenous route was well tolerated byLXFA-737 tumor-bearing mice, showed anti-tumor efficacy (minimum T/C,4.2% on day 27), and its Tq was 80.1%. However, eribulin induced slightand transient bodyweight loss after administration (FIG. 22A and FIG.22B). A single dose of 0.2 mg/kg, double the molar amount of eribulin inMORAb-109 when administered at 10 mg/kg, showed limited anti-tumorefficacy (minimum T/C, 51.1% on day 44).

Example 9: Dose-Response of MORAb-109 in Human Gastric Cancer (NCI-N87)Xenograft Model

9.1 Methods

9.1.1 In Vivo Efficacy

Animals: Female NCr nude mice (Taconic), 5 weeks old at the time ofarrival were acclimated for 5-7 days prior to inoculation. Animals washoused 3-5 mice per ventilated cage with sterilized food pellets andwater bottle available ad lib. Animals were ear tagged and weighed priorto study initiation.

Cell culture: Cryopreserved NCI-N87 cells were thawed and grown inmedium containing necessary supplements. Cells were sub-cultured incomplete medium for 2 passages before being used for in vivoinoculation.

Tumor implantation, enrollment process, and treatment: The cellsuspension in PBS was mixed with ice-cold Matrigel at 1:1 (vol:vol) to afinal concentration of 1.0×10⁸ cells/mL. 100 μL/mouse of the mixture wasinjected subcutaneously. The mice were monitored for clinical well-beingwith body weights and tumors measurements by digital caliper, 3 timesweekly, beginning on day 3 post-implantation.

Tumor measurement and treatment: Tumor volume (TV) (mm³) was calculatedusing the formula: W (mm)×L (mm)×D (mm)×n/6. When the tumors reachedaround 100 mm³ in an average, the animals were randomized to 5 pergroup. Treatment was given intravenously in a volume of 200 μL of testarticle. At the end of the study, the terminal body weight was measuredand recorded.

Statistical analysis: Descriptive statistics were performed on the dataof tumor volume and body weight. Tumor volumes of animals from eachtreatment group were compared with the control group by using therepeated-measures two-way ANOVA followed by the Bonferroni post-test.Additionally, the comparison of tumor growth of animals within eachgroup was performed with the same statistical analysis.

9.1.2 Pharmacokinetics (PK)

PK analysis was performed using both an intact ADC assay and a totalantibody assay. Total antibody refers to the sum total of all species,including conjugated and unconjugated species (i.e., DAR0+DAR1+DAR2+ . .. +DARn), whereas intact ADC refers to all conjugated species (i.e.,DAR1+DAR2+ . . . +DARn). Total antibody assay used biotinylatedmesothelin for capture and AlexaFluor647-labelled anti-human Fc fordetection. Intact ADC assay used biotinylated anti-eribulin 5E4 forcapture and AlexaFluor647-labelled anti-human Fc for detection. Sampleanalysis was performed on a Gyros analyzer. Data analysis was performedin WatsonLIMS 7.4.1 and plotted in Microsoft Excel. Eribulin wasquantitated using LC-MS from plasma, tumor, and bone marrow samples.

9.1.3 LC-MS

20-50 μL of MORAb-109 (DAR2) plasma from individual mice or 50 μL ofequally pooled plasma from MORAb-109 (DAR6)-dosed mice was used foranalysis. Dynabeads M-280 streptavidin (100 μL) were incubated with 3 μgof capture select human IgG-Fc PK biotin conjugate for 1 hour at roomtemperature, then washed with HBS-EP buffer. Plasma samples diluted inHBS-EP buffer were then mixed with the complexed/washed beads forcapture of MORAb-109 complexes, incubated for 1 hour at roomtemperature, then washed twice in HBS-EP buffer. Washed beads containingcomplex were deglycosylated with Rapid PNGaseF (1 μL) in PNGase bufferfor 1 hour at 37° C., then washed once in HBS-EP buffer. Elution ofcaptured/deglycosylated MORAb-109 was done with 10% acetonitrile w/0.1%formic acid (30 μL). Samples were analyzed for intact or reduced masswith Synapt G2/M-class UPLC analysis.

9.2 Results

The anti-tumor effect and body weight change in a human gastric cancerNCI-N87 xenograft model treated with a single dose of MORAb-109 (DAR2 orDAR6) at 10 mg/kg are shown in FIG. 23A and FIG. 23B, respectively.

PK analysis of MORAb-109 (DAR2), MORAb-109 (DAR6), and unconjugatedantibody is shown in Table 27. Total antibody for unconjugated andMORAb-109 (DAR2) was similar, while MORAb-109 (DAR6) was lower,indicating that MORAb-109 (DAR2) is stable in circulation.

DAR analysis of ADCs from plasma samples indicated that the payloadrelease rate was higher for the DAR6 species and contributed to higherplasma levels of eribulin (Tables 28 and 29).

TABLE 27 PK profile of MORAb-109 (DAR2 and DAR6) inNCI-N87-tumor-bearing mice Parameters Unit ADC Total Ab Plasma ERI TumorERI Bone marrow ERI DAR2 CL_(tot) mL/hr/kg 0.71 N/A N/A N/A N/A V_(d)ssmL/kg 121 N/A N/A N/A N/A AUC₀-inf mg × hr/mL 14.2 20.9 44.0 (ng ×hr/mL) 17.8 (μg × hr/mL) 9.9 (μg × hr/mL) T_(max) hr N/A 0.17    0.17 24 1 C_(max) μg/mL N/A 137 0.65 (ng/mL) 64 (ng/g) 121 (ng/g) T_(1/2) hr134 191 59 162  151 DAR6 CL_(tot) mL/hr/kg 0.73 N/A N/A N/A N/A V_(d)ssmL/kg 100 N/A N/A N/A N/A AUC₀-inf mg × hr/mL 13.7 14.4 121 (ng × hr/mL)16.0 (μg × hr/mL) 6.7 (μg × hr/mL) T_(max) hr N/A 0.17  1 24  3 C_(max)μg/mL N/A 148 2.6 (ng/mL) 96 (ng/g) 94 (ng/g) T_(1/2) hr 103 129 33 98 76 Naked Ab CL_(tot) mL/hr/kg 0.54 V_(d)ss mL/kg 126 AUC₀-inf mg ×hr/mL 18.5 C_(max) μg/mL 183 T_(1/2) hr 164

TABLE 28 DAR of MORAb-109 (DAR2) in plasma Averaged Hours 1 2 3 DAR0.167 1.92 1.93 1.94 1.93 8 1.88 1.86 1.92 1.89 24 1.83 1.86 1.90 1.8648 1.87 1.82 1.83 1.84 96 1.83 1.65 1.78 1.75 168 1.69 1.75 1.63 1.69336 1.38 1.54 1.64 1.52 504 1.08 1.18 1.63 1.30

TABLE 29 DAR of MORAb-109 (DAR6) in plasma Averaged Time (hr) L-chainH-chain DAR 0.167 1.52 1.23 5.52 8 1.46 1.13 5.18 24 1.26 0.81 4.15 481.25 0.71 3.91 96 1.27 0.44 3.42 168 0.93 0.38 2.61 336 0.86 0.35 2.41504 N.D. N.D. N.A.

Example 10: Comparison of 345A12 HC15 LC4 with Anetumab—Binding Affinity

10.1 Methods

Antibodies: 345A12 HC15 LC4 (the anti-mesothelin antibody in MORAb-109)and anetumab. Sequences for anetumab, a human anti-mesothelin antibody,are set forth in Table 30.

Binding affinity: Binding measurements were performed in HBS-P+ bufferon a BIAcore T-100 instrument. Antibodies were diluted to 1 μg/mL inHBS-P+. Samples were centrifuged at 14,000×g for 5 min at roomtemperature and then supernatants were transferred to a new 1.5 mLBIAcore tube and capped. Mesothelin (50 μg) was diluted to 100 nM in1×HBS-P+ buffer, then serial diluted 5-fold at 100, 20, 4, 0.8, and 0.16nM in BIAcore tubes and capped. Anti-human antibody capture chip wasprepared according to the manufacturer's protocol using a CMS chip withimmobilization wizard. Final capture antibody levels were 8000-9000RU,in HBS-P+. Chip was prepared for assay with five cycles of 300 secbuffer injection followed by 30 sec regeneration, all at 304/min acrossall four flow cells.

Antibodies were captured on flow cells 2-4 by sequential injections ofindividual ligand solutions for 90 sec at 10 μL/min. Analyte injectionwas done in a single-cycle kinetics manner by sequential injections ofanalyte solutions from low to high concentration for 240 sec each at 30μL/min. Detection was 2-1, 3-1, 4-1. Double-referencing was performed bya sequence of identical ligand capture injections, followed by 5buffer-only injections for 240 sec each, dissociation for 1800 sec, andregeneration as above. All ligands were analyzed for binding tomesothelin in duplicate. Kinetic analysis was performed usingBIAEvaluations software using a 1:1 Langmuir fitting model. On-rate,off-rate, and affinity constants were averaged from duplicate runs.

10.2 Results

345A12 HC15 LC4 exhibited 40-fold higher affinity than anetumab (Table31). 345A12 HC15 LC4 retained binding affinity to cynomolgus monkeymesothelin, while anetumab did not bind cynomolgus monkey mesothelin.Nether antibody bound rat mesothelin.

TABLE 30 Anetumab sequences IgG SEQ chain NA/AA ID Sequence Heavy Amino  51 QVELVQSGAEVKKPGESLKISCKGSGYSFT chain acidSYWIGWVRQAPGKGLEWMGIIDPGDSRTRY SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Nucleic 52 CAGGTTGAACTGGTTCAGTCTGGCGCCGAA acid GTGAAGAAGCCTGGCGAGAGCCTGAAGATCAGCTGCAAAGGCAGCGGCTACAGCTTCACC AGCTATTGGATCGGCTGGGTTCGACAGGCCCCTGGCAAAGGACTGGAATGGATGGGAATC ATCGACCCCGGCGACAGCAGAACCAGATACAGCCCTAGCTTTCAGGGCCAAGTGACCATC AGCGCCGACAAGAGCATCAGCACAGCCTACCTGCAGTGGTCTAGCCTGAAAGCCAGCGAC ACCGCCATGTACTATTGTGCCAGAGGCCAGCTGTACGGCGGCACCTATATGGATGGATGG GGCCAGGGCACACTGGTCACAGTGTCTAGCGCCTCTACAAAGGGCCCTAGCGTTTTCCCA CTGGCTCCTAGCAGCAAGAGCACATCTGGTGGAACAGCCGCTCTGGGCTGCCTGGTCAAG GATTACTTTCCTGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCACTGACAAGCGGCGTG CACACATTTCCAGCTGTGCTGCAGAGCAGCGGCCTGTACTCTCTGTCTAGCGTCGTGACA GTGCCTAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTAGC AACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCT CCATGTCCTGCTCCAGAACTGCTCGGCGGACCCTCCGTTTTCCTGTTTCCACCTAAGCCT AAGGACACCCTGATGATCAGCAGGACCCCTGAAGTGACCTGTGTGGTGGTGGATGTGTCC CACGAGGACCCAGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCC AAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACC GTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCC CTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGAGAACCCCAG GTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCTGC CTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCT GAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTAC AGCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCAGCTGTTCTGTG ATGCACGAGGCCCTGCACAACCACTACACCCAGAAAAGCCTGTCTCTGAGCCCCGGCAAA Light  Amino  53DIALTQPASVSGSPGQSITISCTGTSSDIG chain acid GYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYC SSYDIESATPVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT VAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS Nucleic  54GATATTGCTCTGACACAGCCTGCCAGCGTG acid TCCGGATCTCCTGGCCAGAGCATCACAATCAGCTGTACCGGCACAAGCAGCGACATCGGC GGCTACAATAGCGTGTCCTGGTATCAGCAGCACCCCGGAAAGGCCCCTAAGCTGATGATC TACGGCGTGAACAACAGACCCAGCGGCGTGTCCAATAGATTCAGCGGCAGCAAGAGCGGC AATACCGCCTCTCTGACAATTAGCGGACTGCAGGCCGAGGACGAGGCCGATTACTACTGC AGCAGCTACGACATCGAGAGCGCCACACCTGTGTTTGGCGGCGGAACAAAACTGACAGTG CTGGGCCAACCTAAGGCCGCTCCTAGCGTTACACTGTTCCCACCTAGCAGCGAGGAACTG CAGGCTAACAAGGCCACACTCGTGTGCCTGATCAGCGATTTTTACCCTGGCGCCGTGACA GTGGCCTGGAAAGGCGATAGTTCTCCTGTGAAGGCCGGCGTGGAAACCACCACACCTAGC AAGCAGAGCAACAACAAATACGCCGCCAGCTCCTACCTGAGCCTGACACCTGAGCAGTGG AAGTCCCACAGATCCTACAGCTGCCAAGTGACCCACGAGGGCAGCACCGTGGAAAAAACA GTGGCCCCTACCGAGTGCAGC

TABLE 31 Binding of 345A12 HC15 LC4 and anetumab to human, cynomologusmonkey, and rat mesothelin k_(a) (M⁻¹sec⁻¹) k_(d) (sec⁻¹) K_(D) (M)human 345A12 1.46 ± 2.85 ± 1.95 ± HC15 LC4 0.05 × 10⁶ 0.14 × 10⁻⁴ 0.16 ×10⁻¹⁰ anetumab 1.68 ± 1.12 ± 7.26 ± 0.64 × 10⁵ 1.00 × 10⁻³ 2.29 × 10⁻⁹cynomolgus 345A12 1.05 ± 7.51 ± 8.86 ± HC15 LC4 1.15 × 10⁴ 5.76 × 10⁻⁴5.16 × 10⁻⁸ anetumab no no no binding binding binding 345A12 no no noHC15 LC4 binding binding binding rat anetumab no no no binding bindingbinding

Example 11: Comparison of MORAb-109 with BAY 94-9343—In Vitro Potency

11.1 Methods

ADCs: MORAb-109 (DAR2 and DAR6) and anetumab ravtansine (BAY 94-9343)were evaluated. Anetumab ravtansine, also referred to as BAY 94-9343, isan ADC comprising anetumab conjugated to the maytansinoid tubulininhibitor DM4 via a disulfide-containing linker (a reducible SPDB linker[N-succinimidyl 4-(2-pyridyldithio)butanoate]). BAY 94-9343 wasgenerated as described in Example 15.

Cytotoxicity: Cells were sub-cultured and seeded at 5,000 cells/well incomplete growth medium in 96-well tissue culture plates, and incubatedat 37° C., 5% CO₂ overnight (16 hours). Test reagents were seriallydiluted 1:3 in 2 mL deep-well dilution plates, starting at 200 nM (10dilutions total). Diluted samples (100 μL) were added to the cell plates(starting concentration of test samples at 100 nM). Plates wereincubated at 37° C., 5% CO₂ for an additional 5 days. Medium was thendiscarded, and plates were washed once with 200 μL DPBS, stained with 50μL of 0.2% Crystal Violet solution at room temperature for 15 min, andthen washed extensively with tap water. Plates were air-dried, andCrystal Violet was dissolved with 200 μL of 1% SDS solution. Plates wereread at 570 nm. Data was analyzed for IC₅₀ determination using GraphPadPrism 6.

11.2 Results

MORAb-109 (DAR2 and DAR6) showed specific cytotoxicity on MSLN-positivecell lines (Table 32). BAY 94-9343, in contrast, demonstrated killing onboth MSLN-positive and MSLN-negative cell lines.

TABLE 32 Comparison of in vitro activity on MSLN⁺ and MSLN⁻ cell linesOVCAR3-A1 NCI-H1568/MSLN NCI-H292 BxPC3 A431 SKOV3 In vitro cell basedpotency, IC50, nM MORAb109_DAR2 0.01 2.17 >300 86.24 109.30 213.80MORAb109_DAR6 0.02 1.09 4.80 2.66 20.12 21.77 Eribulin 0.02 0.14 0.110.13 0.18 0.14 BAY 94-9343 0.41 0.15 3.07 2.53 5.31 0.81 DM4 0.10 0.570.24 0.57 0.71 0.28 anetumab >100 >100 >300 >300 >300 >300 % Max KillingMORAb109_DAR2 98.39 73.49 56.22 90.62 84.97 58.41 MORAb109_DAR6 98.4777.89 75.34 94.63 90.76 72.79 Eribulin 99.31 74.39 85.37 97.28 93.0472.82 BAY 94-9343 100.00 75.10 64.15 97.33 96.82 74.99 DM4 100.00 77.4992.40 97.98 92.52 81.89 anetumab 19.80 5.50 15.15 25.73 18.58 31.55 MSLNexpression (MFI) 220.0 4474.00 108.6 15.59 5.6 20

Example 12: Comparison of MORAb-109 with BAY 94-9343—Specificity

12.1 Methods

Cytotoxicity: Cells were sub-cultured and seeded at 5,000 cells/well incomplete growth medium in 96-well tissue culture plates, and incubatedat 37° C., 5% CO₂ overnight (16 hours). Test reagents were seriallydiluted 1:3 in 2 mL deep-well dilution plates, starting at 200 nM (10dilutions total). Diluted samples (100 μL) were added to the cell plates(starting concentration of test samples at 100 nM). Plates wereincubated at 37° C., 5% CO₂ for an additional 5 days. Medium was thendiscarded, and plates were washed once with 200 μL DPBS, stained with 50μL of 0.2% Crystal Violet solution at room temperature for 15 min, andthen washed extensively with tap water. Plates were air-dried, andCrystal Violet was dissolved with 200 μL of 1% SDS solution. Plates wereread at 570 nm. Data was analyzed for IC₅₀ determination using GraphPadPrism 6.

12.2 Results

Cytotoxicity assays with unconjugated antibody demonstrated specifickilling of mesothelin-expressing cells by MORAb-109 (DAR2) (FIG. 25A),but not BAY 94-9343 (FIG. 25B). Without being bound by theory, the lackof competition by unconjugated antibody observed for BAY 94-9343suggests release of payload, which can lead to killing even whenantibody binding is blocked by an unconjugated competitor. This payloadrelease is consistent with the relatively high levels of cytotoxicityobserved for BAY 94-9343 on mesothelin-negative cell lines (Table 32).Payload release is also directly observed in FIG. 27 (plasma stabilitycomparison).

Example 13: Comparison of MORAb-109 with BAY 94-9343—ADCC Activity

13.1 Methods

MSLN-expressing CHO cells were thawed and seeded 1,000 cells/well (25μL) in 96-well tissue culture plates in complete RPMI-4% Ultralow IgGFBS. Test reagents (345A12 antibody, MORAb-109 (DAR2), and BAY 94-9343)were 1:2.5 serial diluted starting from 20 μg/mL in complete RPMI-4%ultra-low IgG FBS, then transferred (25 μL) to the cell plate, andincubated at 37° C., 5% CO₂ for 60 min. 6,000 Jurkat-Effector cells(Promega) were thawed and added (25 μL) to the cell plate, and the platewas incubated at 37° C., 5% CO₂ for 18-22 hours.

Luciferase assay reagent was thawed in the dark. 75 μL of luciferaseassay reagent was added to each well, plates were shaken for 30 sec on aplate shaker. Plates were read on a luminometer after 5 min incubation.

13.2 Results

MORAb-109 (DAR2) and 345A12 HC15 LC4 had similar ADCC activity (FIG. 26Aand Table 33), while BAY 94-9343 had weaker ADCC activity than anetumab(FIG. 26B and Table 34).

TABLE 33 ADCC activity - MORAb-109 and 345A12 HC15 LC4 345A12 MORAb-109100% 96.9%

TABLE 34 ADCC activity - BAY 94-9343 and anetumab anetumab BAY 94-9343100% 65.06%

Example 14: Stability of MORAb-109 and BAY 94-9343 in Matrix

14.1 Methods

Anti-MSLN ADCs were prepared at 0.1 mg/mL either in human or mouseplasma, the samples were incubated at 37° C. for 0, 24, 48, 72, 96 and240 hours, then transferred to −80° C. for storage when timepoints wereachieved. All samples were thawed to ambient temperature and diluted1:100 for testing. A DAR-sensitive stability assay was developed asstepwise sandwich format on Gyrolab. Assay used biotinylated mesothelinfor capture after blocking and sample binding, and Alexa Fluor 647anti-eribulin 5E4 Fab or Alexa Fluor 647 anti-DM4 (Levena Biopharma) fordetection. Standard curve and quality controls were made with MORAb-109and BAY 94-9343.

14.2 Results

MORAb-109 (DAR2) was more stable than BAY 94-9343 in both human andmouse plasma (FIG. 27 ).

Example 15: Anti-Tumor Efficacy of MORAb-109 and BAY 94-9343 in HumanGastric Cancer (NCI-N87) Xenograft Model

15.1 Methods

15.1.1 Generation of BAY 94-9343

BAY 94-9343 is an ADC comprising anetumab conjugated to the maytansinoidtubulin inhibitor DM4 via a disulfide-containing linker (a reducibleSPDB linker [N-succinimidyl 4-(2-pyridyldithio)butanoate]). Sequencesfrom anetumab were obtained from Beacon database (Hanson-Wade). Antibodysequences (Table 30) were generated from overlapping oligonucleotides,PCR-amplified, cloned into expression plasmids, and sequence-confirmed.Stable pools were generated in 293F cells and cells were grown untilviability <30%. Anetumab was purified from conditioned medium usingprotein A affinity chromatography. BAY 94-9343 was generated bylysine-reactive conjugation with SPDB-DM4 (Levena BioPharma) to achievea DAR of 3.7. Unconjugated payload was removed by desaltingchromatography.

15.1.2 In Vivo Efficacy

Animals: Female NCr nude mice (Taconic), 5 weeks old at the time ofarrival were acclimated for 5-7 days prior to inoculation. Animals washoused 3-5 mice per ventilated cage with sterilized food pellets andwater bottle available ad lib. Animals were ear tagged and weighed priorto study initiation.

Cell culture: Cryopreserved NCI-N87 cells were thawed and grown inmedium containing necessary supplements. Cells were sub-cultured incomplete medium for 2 passages before being used for in vivoinoculation.

Tumor implantation, enrollment process, and treatment: The cellsuspension in PBS were mixed with ice-cold Matrigel at 1:1 (vol:vol) toa final concentration of 1.0×10⁸ cells/mL. 100 μL/mouse of the mixturewas injected subcutaneously. The mice were monitored for clinicalwell-being with body weights and tumors measurements by digital caliper,3 times weekly, beginning on day 3 post-implantation.

Tumor measurement and treatment: Tumor volume (TV) (mm³) was calculatedusing the formula: W (mm)×L (mm)×D (mm)×n/6. When the tumors reachedaround 100 mm³ in an average, the animals were randomized to 5 pergroup. Treatment was given intravenously in a volume of 200 μL of testarticle. At the end of the study, the terminal body weight was measuredand recorded.

Statistical analysis: Descriptive statistics were performed on the dataof tumor volume and body weight. Tumor volumes of animals from eachtreatment group were compared with the control group by using therepeated-measures two-way ANOVA followed by the Bonferroni post-test.Additionally, the comparison of tumor growth of animals within eachgroup was performed with the same statistical analysis.

15.2 Results

MORAb-109 (DAR2) and BAY 94-9343 both demonstrated similar efficacy inNCI-N87 tumor-bearing mice (FIG. 28A). No body weight loss was observedin either group (FIG. 28B).

Example 16: Anti-Tumor Efficacy of MORAb-109 and BAY 94-9343 in HumanMesothelioma (HAY) Xenograft Model

16.1 Methods

Animals: Female NOD.CB17-SCID mice (Jackson Laboratory), 5 weeks old atthe time of arrival were acclimated for 5-7 days prior to inoculation.Animals was housed 3-5 mice per ventilated cage with sterilized foodpellets and water bottle available ad lib. Animals were ear tagged andweighed prior to study initiation.

Cell culture: Cryopreserved HAY cells were thawed and grown in mediumcontaining necessary supplements. Cells were sub-cultured in completemedium for 2 passages before being used for in vivo inoculation.

Tumor implantation, enrollment process, and treatment: The cellsuspension in PBS were mixed with ice-cold Matrigel at 1:1 (vol:vol) toa final concentration of 5.0×10⁷ cells/mL. 100 μL/mouse of the mixturewas injected subcutaneously. The mice were monitored for clinicalwell-being with body weights and tumors measurements by digital caliper,3 times weekly, beginning on day 3 post-implantation.

Tumor measurement and treatment: Tumor volume (TV) (mm³) was calculatedusing the formula: W (mm)×L (mm)×D (mm)×n/6. When the tumors reachedaround 100 mm³ in an average, the animals were randomized to 5 pergroup. Treatment was given intravenously in a volume of 200 μL of testarticle. At the end of the study, the terminal body weight was measuredand recorded.

Statistical analysis: Descriptive statistics were performed on the dataof tumor volume and body weight. Tumor volumes of animals from eachtreatment group were compared with the control group by using therepeated-measures two-way ANOVA followed by the Bonferroni post-test.Additionally, the comparison of tumor growth of animals within eachgroup was performed with the same statistical analysis.

16.2 Results

MORAb-109 (DAR2) and BAY 94-9343 both demonstrated similar efficacy inHAY tumor-bearing mice (FIG. 29A). No body weight loss was observed ineither group (FIG. 29B).

Example 17: Anti-Tumor Efficacy of MORAb-109 and BAY 94-9343 in HumanMesothelioma PDX Model (Meso7212)

17.1 Methods

Animals: NMRI nu/nu female mice (Janvier Labs), 5 to 6 weeks old at thetime of arrival, were acclimated for at least 4 days prior toinoculation. Animals were housed 3-5 mice per ventilated cage withsterilized food pellets and water bottle available ad lib. Animals wereear marked and weighed prior to study initiation

Xenotransplantation: On day 0, Meso 7212 tumors were removed from thefive donor mice under sterile conditions. The tumor tissue was cut into2×2 mm fragments and placed in a sterile Petri dish covered with 0.9%saline. In parallel, the receptor animals were subcutaneouslyanalgesic-treated with Metacam® (2 mg/kg) and then anaesthetized by asingle intravenous injection (0.15 mL/mouse) with Etomidat-Lipuro® (12mg/kg). A superficial vertical incision in the skin of 5-8 mm on theleft flank was performed. The tip of a surgical scissor was insertedinto the incision directly over the flank and was used to form a pocketin the subcutaneous space. One tumor fragment per mouse was implantedinto the pocket using surgical tweezers. Finally, the incision wasclosed with a metal clip and the animal placed in a clean cage.

Experimental procedure: After the xenotransplantation, the engraftmentand the propagation of the tumor in the mice were controlled at leasttwice weekly by palpation. When the tumor was palpable, the measurementsof tumor diameters were performed with a digital caliper (Mitutoyo).

Prior starting the treatment, animals were randomly assigned into theexperimental groups according to their tumor volume (inclusion criteriafor tumor volume, 0.1-0.3 cm³). From the first treatment day onwards,tumor volumes and body weights were recorded twice weekly. The animalwelfare was controlled twice daily.

Treatment: All agents were administered intravenously as a single doseon the day of randomization. Animals in the control group were treatedwith DPBS in the same manner.

Statistical analysis: Descriptive statistics were performed on the dataof tumor volume and body weight. Tumor volumes of animals from eachtreatment group were compared with the control group by using therepeated-measures two-way ANOVA followed by the Bonferroni post-test.Additionally, the comparison of tumor growth of animals within eachgroup was also performed with the same statistical analysis.

17.2 Results

MORAb-109 (DAR2) and BAY 94-9343 both demonstrated tumor regression inMeso7212 tumor-bearing mice (FIG. 30A). No body weight loss was observedin either group (FIG. 30B).

Example 18: Anti-Tumor Efficacy of MORAb-109 and BAY 94-9343 in HumanNSCLC PDX Model (LXFA-586)

18.1 Methods

Animals: Female NMRI nu/nu mice, 4 to 6 weeks of age.

Xenotransplantation: LXFA-586 established growing tumor (T2N1M0) from aprimary non-small cell lung adenocarcinoma human patient.

Experimental procedure: Animals were monitored until the tumor implantsreached the study volume criteria of 50-250 mm³ (e.g., 80-200 mm³) in asufficient number of animals. Mice were assigned to groups aiming atcomparable group median and mean tumor volumes. The process of theassignment to groups (enrollment, stratified randomization) may also bereferred to as randomization. The day of randomization was designated asday 0 of the experiment.

Treatment: Efficacy was evaluated in 4 groups of 6 to 7 mice each:

-   -   In group 1, vehicle was dosed at 5 mL/kg, i.v., single dose on        day 1.    -   In group 2, BAY 94-9343 (DAR˜4) was dosed at 25 mg/kg, i.v.,        single dose on day 1.    -   In group 3, MORAb-109 (DAR2) was dosed at 25 mg/kg, i.v., single        dose on day 1.    -   In group 4, eribulin was dosed at 3.2 mg/kg, i.v., single dose        on day 1.

Tumors were measured and mice were weighed twice a week during theexperimental period. The first day of dosing was day 1, one day afterrandomization (day 0).

18.2 Results

MORAb-109 (DAR2) demonstrated robust anti-tumor efficacy (minimum T/C,1.8% on day 41) at 25 mg/kg in the LXFA-586 NSCLC PDX model (FIG. 31A)and its Tq was not reached during the study. MORAb-109 given at thesingle dose was also well tolerated without bodyweight loss by LXFA-586tumor-bearing mice (FIG. 31B).

BAY 94-9343 (DAR˜4) demonstrated robust anti-tumor efficacy similar toMORAb-109 at 25 mg/kg in the LXFA-586 NSCLC PDX model (FIG. 31A) and itsTq was not reached during the study. However, the molar amount of DM4payload in BAY 94-9343 is approximately twice the amount of eribulinpayload in MORAb-109.

Eribulin given at a single dose of 3.2 mg/kg (mouse MTD dosage or 32times higher than the molar amount of eribulin in MORAb-109 whenadministered at 10 mg/kg) by intravenous route was well tolerated byLXFA-586 tumor-bearing mice, and showed anti-tumor efficacy (minimumT/C, 14.8% on day 21). However, eribulin induced slight and transientbodyweight loss after administration (FIG. 31A and FIG. 31B).

Example 19: Anti-Tumor Efficacy of MORAb-109 and BAY 94-9343 in HumanNSCLC PDX Model (LXFL-529)

19.1 Methods

Animals: Female NMRI nu/nu mice, 4 to 6 weeks of age.

Xenotransplantation: LXFL-529 established growing tumor (T3N1M0) from aprimary non-small cell lung adenocarcinoma human patient.

Experimental procedure: Animals were monitored until the tumor implantsreached the study volume criteria of 50-250 mm³ (e.g., 80-200 mm³) in asufficient number of animals. Mice were assigned to groups aiming atcomparable group median and mean tumor volumes. The process of theassignment to groups (enrollment, stratified randomization) may also bereferred to as randomization. The day of randomization was designated asday 0 of the experiment.

Treatment: Efficacy was evaluated in 6 groups of 6 to 7 mice each:

-   -   In group 1, vehicle was dosed at 5 mL/kg, i.v., single dose on        day 1.    -   In group 2, BAY 94-9343 (DAR˜4) was dosed at 12.5 mg/kg, i.v.,        single dose on day 1.    -   In group 3, eribulin was dosed at 3.2 mg/kg, i.v., single dose        on day 1.    -   In group 4, MORAb-109 (DAR2) was dosed at 25 mg/kg, i.v., single        dose on day 1.    -   In group 5, MORAb-109 (DAR2) was dosed at 12.5 mg/kg, i.v.,        single dose on day 1.    -   In group 6, MORAb-109 (DAR2) was dosed at 12.5 mg/kg, i.v.,        doses on days 1, 8, and 16.

Tumors were measured and mice were weighed twice a week during theexperimental period. The first day of dosing was day 1, one day afterrandomization (day 0).

19.2 Results

MORAb-109 (DAR2) demonstrated robust anti-tumor efficacy at 12.5 and 25mg/kg in the LXFL-529 NSCLC PDX model (FIG. 32A). MORAb-109 given at thesingle dose was also well tolerated without bodyweight loss by LXFL-529tumor-bearing mice (FIG. 32B).

BAY 94-9343 (DAR˜4), however, at 12.5 mg/kg (the equivalent molar amountof DM4 payload as the amount of eribulin payload in MORAb-109 at 25mg/kg) demonstrated no anti-tumor efficacy (FIG. 32A).

Eribulin given at a single dose of 3.2 mg/kg (mouse MTD dosage) byintravenous route was well tolerated by LXFL-529 tumor-bearing mice, andshowed anti-tumor efficacy. However, eribulin induced slight andtransient bodyweight loss after administration (FIG. 32A and FIG. 32B).

The invention claimed is:
 1. An isolated antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is capable of binding to mesothelin and comprises heavy chain complementarity determining regions comprising amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3); and light chain complementarity determining regions comprising amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or heavy chain complementarity determining regions comprising amino acid sequences of SEQ ID NO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3); and light chain complementarity determining regions comprising amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system.
 2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:
 14. 3. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a light chain comprising the amino acid sequence of SEQ ID NO:
 18. 4. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment is conjugated to a therapeutic agent.
 5. The antibody or antigen-binding fragment of claim 4, wherein the therapeutic agent is eribulin.
 6. A pharmaceutical composition comprising the antibody or antigen-binding fragment of claim 1, and a pharmaceutically acceptable carrier. 